Methods related to TIM 3, a Th1-specific cell surface molecule, for activating antigen presenting cells

ABSTRACT

The present invention provides compositions and methods useful for promoting or reducing T-cell trafficking to a target tissue. Also provided are compositions and methods useful for promoting or inhibiting antigen-presenting cell (APC) activation. The invention is related to discovery of functional characteristics of TIM-3, a molecule that is preferentially expressed on the surface of Th1 cells. The methods are useful for treating disorders including cancer, infectious disease, allergy, asthma, and autoimmune disease.

CROSS-REFERENCED APPLICATIONS

This application is a Continuation application that claims benefit under35 U.S.C. §120 of U.S. application Ser. No. 14/185,050, filed Feb. 20,2014, which is a Continuation application that claims benefit under 35U.S.C. §120 of U.S. application Ser. No. 13/331,387, filed Dec. 20,2011, now U.S. Pat. No. 8,697,069, which is a Continuation applicationthat claims benefit under 35 U.S.C. §120 of U.S. application Ser. No.12/276,977 filed Nov. 24, 2008, now U.S. Pat. No. 8,101,176, which is aContinuation application that claims benefit under 35 U.S.C. §120 ofU.S. application Ser. No. 10/354,447 filed Jan. 30, 2003, now U.S. Pat.No. 7,470,428, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 60/353,107, filed Jan. 30, 2002, thecontents of each of which are incorporated herein by reference in theirentireties.

GOVERNMENT RIGHTS

This invention was funded in part under National Institutes of HealthGrant Nos. R01-NS30843 and R01-NS38037. The United States government mayretain certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 30, 2003, isnamed Seqlist.txt and is 17,776 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methodsuseful for regulating the immune response. More particularly, theinvention relates to methods of promoting and inhibiting immune effectorcell function at the level of T-cell trafficking to target tissues andmacrophage activation, both of which are disclosed herein to be relatedto functions of the Th1-specific cell surface molecule T cellImmunoglobulin and Mucin domain-containing molecule-3 (TIM-3). Theinvention also relates to methods of treating disorders such as cancer,infectious disease, allergy, asthma, and autoimmune disease.

BACKGROUND OF THE INVENTION

Activation of naïve CD4+ T helper cells results in the development of atleast two distinct effector populations, Th1 cells and Th2 cells.Mosmann T R et al. (1986) J Immunol 136:2348-57; Mosmann T R et al.(1996) Immunol Today 17:138-46; Abbas A K et al. (1996) Nature383:787-793. Th1 cells produce cytokines (interferon gamma (IFN-γ),interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), andlymphotoxin) which are commonly associated with cell-mediated immuneresponses against intracellular pathogens, delayed-type hypersensitivityreactions (Sher A et al. (1992) Annu Rev Immunol 10:385-409), andinduction of organ-specific autoimmune diseases. Liblau R S et al.(1995) Immunol Today 16:34-38. Th2 cells produce cytokines (IL-4, IL-10,and IL-13) that are crucial for control of extracellular helminthicinfections and promote atopic and allergic diseases. Sher A et al.(1992) Annu Rev Immunol 10:385-409. In addition to their distinct rolesin disease, the Th1 and Th2 cells cross-regulate each other's expansionand functions. Thus, preferential induction of Th2 cells inhibitsautoimmune diseases (Kuchroo V K et al. (1995) Cell 80:707-18; NicholsonL B et al. (1995) Immunity 3:397-405), and predominant induction of Th1cells can regulate induction of asthma, atopy and allergies. Lack G etal. (1994) J Immunol 152:2546-54; Hofstra C L et al. (1998) J Immunol161:5054-60.

While much is known about the functions of these T-cell subsets, thereare few known surface molecules that distinguish between them. Syrbe Uet al. (1999) Springer Semin Immunopathol 21:263-85. Several groups havereported the association of certain chemokine and costimulatory moleculereceptors with Th1 cells. Loetscher P et al. (1998) Nature 391:344-45;Bonecchi R et. al. (1998) J Exp Med 187:129-34; Sallusto F et al. (1998)J Exp Med 187:875-83; Venkataraman C et al. (2000) J Immunol 165:632-36.Likewise, several groups have reported the association of certainchemokine and costimulatory molecule receptors with Th2 cells. BonecchiR et al. (1998) J Exp Med 187:129-34; Sallusto F et al. (1998) J Exp Med187:875-83; Jourdan P et al. (1998) J Immunol 160:4153-57; Zingoni A etal. (1998) J Immunol 161:547-51; McAdam A J et al. (2000) J Immunol165:5035-40; Lohning M et al. (1998) Proc Natl Acad Sci USA 95:6930-35.However, the nature of the differences in expression of most of thesemolecules is quantitative.

U.S. Pat. No. 6,084,083, issued to Levinson discloses a murineTh1-restricted cell surface molecule termed the “200 gene product,”along with its human homolog. The murine 200 gene product is theredisclosed as a 280-amino acid membrane-bound member of theimmunoglobulin (Ig) superfamily. The human homolog of the murine 200gene product is there disclosed as a 301-amino acid membrane-boundmember of the immunoglobulin (Ig) superfamily. Full-length nucleotideand amino acid sequences of the murine and human forms of the 200 geneand the 200 gene product, antibodies specific for the 200 gene product,and soluble forms of the 200 gene product are disclosed. Despite itsidentification as a Th1-restricted cell surface molecule, the functionof the 200 gene and the endogenous ligand of the 200 gene are notdisclosed in U.S. Pat. No. 6,084,083.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification andstructural and functional characterization of a transmembrane protein,TIM-3, which is preferentially expressed on differentiated Th1 cells.Full-length nucleotide and amino acid sequences of both human and murineforms of TIM-3 are disclosed. Comparison of these sequences withcorresponding sequences of the independently discovered 200 genes and200 gene products disclosed in U.S. Pat. No. 6,084,083 reveals theiridentity. Surprisingly, however, in vivo administration of antibody toTIM-3 enhances the clinical and pathologic severity of experimentalautoimmune encephalomyelitis (EAE), a Th1-dependent autoimmune diseasethat is widely accepted as a model for multiple sclerosis, ademyelinating disorder in humans. In vivo administration of antibody toTIM-3 also increases the number and activation level of macrophages.TIM-3 may play an important role in the induction of autoimmune diseasesby regulating macrophage activation and/or function. Thus TIM-3 plays animportant role in the activation and expansion of macrophages inperipheral lymphoid tissue. As further disclosed herein, cognateinteraction between T cells and macrophages is involved in thismacrophage expansion and activation. The expansion and activation ofmacrophages is directed by the Th1 cells and is TIM-3-dependent.

The invention is also based in part on the unexpected finding that TIM-3expression on effector Th1 cells promotes migration of Th1 cells totarget tissues to mediate inflammation and immune response. As furtherdisclosed herein, TIM-3-dependent trafficking of effector Th1 cells canbe augmented with antibody to TIM-3 and inhibited with a soluble form ofTIM-3.

Full-length TIM-3 is believed to be expressed as a membrane-associatedprotein having an extracellular region including an IgV domain and amucin domain, a transmembrane region, and a cytoplasmic region. Theinvention is also based in part on the surprising discovery by theinventors of an alternatively spliced variant of TIM-3 in which themucin domain and transmembrane region are deleted. This alternativelyspliced variant of TIM-3 is believed to represent a naturally occurringform of soluble TIM-3.

In one aspect of the invention, a monoclonal antibody 8B.2C12 that bindsspecifically to TIM-3 is provided. Also provided in another aspect ofthe invention is a hybridoma 8B.2C12 that expresses the monoclonalantibody 8B.2C12.

In another aspect of the invention, the invention provides a monoclonalantibody 25F.1D6 that binds specifically to TIM-3. Also providedaccording to another aspect of the invention is a hybridoma 25F.1D6 thatexpresses the monoclonal antibody 25F.1D6.

In other aspects the invention provides pharmaceutical compositionscontaining the foregoing monoclonal antibodies specific for TIM-3. Inone aspect the pharmaceutical composition includes monoclonal antibody8B.2C12 and a pharmaceutically acceptable carrier. In another aspect thepharmaceutical composition includes monoclonal antibody 25F.1D6 and apharmaceutically acceptable carrier.

Also provided are methods for preparing the pharmaceutical compositionscontaining the foregoing monoclonal antibodies specific for TIM-3. Inone aspect the invention provides a method for preparing apharmaceutical composition. The method involves placing monoclonalantibody 8B.2C12 in a pharmaceutically acceptable carrier. In anotheraspect the invention provides a method for preparing a pharmaceuticalcomposition. The method involves placing monoclonal antibody 25F.1D6 ina pharmaceutically acceptable carrier.

In another aspect the invention provides a method for treating a subjectin need of an enhanced immune response in a target tissue. The methodinvolves administering to the subject a TIM-3-binding molecule in aneffective amount to promote T-cell trafficking to the target tissue. Insome embodiments the TIM-3-binding molecule is an antibody specific forTIM-3. In some embodiments the TIM-3-binding molecule is a fragment ofan antibody specific for TIM-3.

In one embodiment the TIM-3-binding molecule is an antibody expressed byhybridoma 8B.2C12. In another embodiment the TIM-3-binding molecule isan antibody expressed by hybridoma 25F.1D6.

In some embodiments the TIM-3-binding molecule binds to an extracellularregion of TIM-3. In accordance with the structure of TIM-3 as disclosedherein, in some embodiments the extracellular region of TIM-3 is an IgVdomain or a fragment thereof, and in some embodiments the extracellularregion of TIM-3 is a mucin domain or a fragment thereof.

In some preferred embodiments the subject has cancer or is at risk ofhaving cancer. In some preferred embodiments the subject has aninfection or is at risk of having an infection.

In some embodiments according to this aspect of the invention, thetarget tissue is selected from the group consisting of: brain, breast,lung, kidney, liver, pancreas, stomach, intestine, ovary, uterus,testis, prostate, marrow, bone, muscle, and skin. In one preferredembodiment, the target tissue is central nervous system.

In a preferred embodiment the subject is a human.

Also according to this aspect of the invention, in some embodiments theadministering is to a site other than the target tissue. In someembodiments the administering is to a site other than a lymph nodeassociated with the target tissue.

In some embodiments the administering is systemic. In a preferredembodiment the administering is intravenous.

Also according to this aspect of the invention, in some embodiments themethod further entails administering to the subject an adjuvant.

In some embodiments the method according to this aspect of the inventionfurther involves administering to the subject an anti-tumor medicament.In some preferred embodiments the anti-tumor medicament includes atumor-specific antibody or tumor-specific fragment thereof.

Other agents can be administered as part of the method according to thisaspect. For example, in some embodiments the method also includesadministering to the subject a cytokine. In some embodiments the methodaccording to this aspect of the invention further involves administeringto the subject an antibacterial medicament. In some embodiments themethod further involves administering to the subject an antiviralmedicament. In some embodiments the method according to this aspect ofthe invention further involves administering to the subject anantifungal medicament. In some embodiments the method further involvesadministering to the subject an antiparasitic medicament.

In another aspect the invention provides a method for treating a subjectin need of treatment for a tumor. The method according to this aspectinvolves administering to the subject a TIM-3-binding molecule in aneffective amount to promote T-cell trafficking to the tumor.

In one embodiment the TIM-3-binding molecule is an antibody expressed byhybridoma 8B.2C12. In another embodiment the TIM-3-binding molecule isan antibody expressed by hybridoma 25F.1D6.

In another aspect the invention provides a method for treating a subjectin need of treatment for an infection. The method according to thisaspect involves administering to the subject a TIM-3-binding molecule inan effective amount to promote T-cell trafficking to the infection.

In one embodiment the TIM-3-binding molecule is an antibody expressed byhybridoma 8B.2C12. In another embodiment the TIM-3-binding molecule isan antibody expressed by hybridoma 25F.1D6.

In another aspect the invention provides a method for reducing T-celltrafficking into a target tissue of a subject. The method according tothis aspect involves administering to the subject a TIM-3 ligand-bindingmolecule in an effective amount to reduce T-cell trafficking to a targettissue of the subject. In some embodiments the TIM-3 ligand-bindingmolecule includes at least one domain of an extracellular region ofTIM-3. In one embodiment the at least one domain is an IgV domain. Insome embodiments the TIM-3 ligand-binding molecule is soluble TIM-3.Preferably the soluble TIM-3 is a fusion protein including at least onedomain of an extracellular region of TIM-3 and a constant heavy chain orportion thereof of an immunoglobulin. In one embodiment the at least onedomain is an IgV domain.

In some embodiments the subject is in need of treatment for anautoimmune disease of the target tissue. In certain preferredembodiments the target tissue is selected from the group consisting of:central nervous system, pancreatic islets, and joint synovia. In certainpreferred embodiments the autoimmune disease is selected from the groupconsisting of: multiple sclerosis, type 1 diabetes mellitus, andrheumatoid arthritis.

In yet another aspect the invention provides a method for treating orpreventing asthma or allergy. The method according to this aspect of theinvention involves increasing activity or expression of TIM-3 in a Tcell of a subject to treat or prevent asthma or allergy. In oneembodiment the T cell is a Th2 cell.

According to a further aspect, the invention provides a method fortreating a Th2-mediated disorder in a subject. The method involvesexpressing TIM-3 on the surface of Th2 cells of a subject having aTh2-mediated disorder in an amount effective to treat the Th2-mediateddisorder. In a preferred embodiment the Th2-mediated disorder is asthma.

In another aspect of the invention, a method is provided for promotingantigen-presenting cell (APC) activation. The method entails contactingan APC with a TIM-3 ligand-binding molecule in an effective amount toactivate the APC.

In some embodiments the APC is a macrophage. In some embodiments the APCis a dendritic cell.

In some embodiments the TIM-3 ligand-binding molecule includes at leastone domain of an extracellular region of TIM-3. In one embodiment the atleast one domain is an IgV domain. In some embodiments the TIM-3ligand-binding molecule is soluble TIM-3. Preferably the soluble TIM-3is a fusion protein including at least one domain of an extracellularregion of TIM-3 and a constant heavy chain or portion thereof of animmunoglobulin. In one embodiment the at least one domain is an IgVdomain.

According to another aspect, the invention provides a method forpromoting APC activation. The method according to this aspect involvescontacting a T cell with a TIM-3-binding molecule, and contacting an APCwith the T cell to activate the APC.

In some embodiments the APC is a macrophage. In some embodiments the APCis a dendritic cell.

In some embodiments the TIM-3-binding molecule is an antibody specificfor TIM-3. In one embodiment the TIM-3-binding molecule is an antibodyexpressed by hybridoma 8B.2C12. In another embodiment the TIM-3-bindingmolecule is an antibody expressed by hybridoma 25F.1D6.

In some embodiments the TIM-3-binding molecule is a fragment of anantibody specific for TIM-3. In some embodiments the TIM-3-bindingmolecule binds to an extracellular region of TIM-3.

In some embodiments the method further includes contacting the T cellwith an antigen specifically bound by a T-cell antigen receptor of the Tcell.

In some embodiments the method further includes contacting the APC withan antibody specific for TIM-3.

In some embodiments according to this aspect of the invention, thecontacting the APC with the T cell is ex vivo.

In one preferred embodiment the antigen is a tumor antigen.

According to yet another aspect of the invention, a method is providedfor inhibiting APC activation. The method involves contacting an APCwith an agent that reduces activity or expression of TIM-3 in aneffective amount to inhibit activation of the APC.

In some embodiments the APC is a macrophage. In some embodiments the APCis a dendritic cell.

In some embodiments the agent that reduces activity or expression ofTIM-3 is soluble TIM-3.

In some embodiments the agent that reduces activity or expression ofTIM-3 includes at least one domain of an extracellular region of TIM-3.In one embodiment the at least one domain is an IgV domain.

In some embodiments the agent that reduces activity or expression ofTIM-3 is a fusion protein including at least one domain of anextracellular region of TIM-3 and a constant heavy chain or portionthereof of an immunoglobulin. In one embodiment the at least one domainis an IgV domain.

In some embodiments the agent that reduces activity or expression ofTIM-3 is an antisense polynucleotide capable of hybridization with anucleic acid encoding TIM-3 under stringent hybridization conditions.

According to another aspect the invention further provides a method fortreating or preventing intracellular infections. The method according tothis aspect involves promoting macrophage activation by contacting aTIM-3 ligand on the macrophage with a TIM-3 expressing cell.

According to yet another aspect the invention provides a method fortreating or preventing cancer. The method according to this aspectinvolves promoting APC activation by contacting a TIM-3 ligand on theAPC with a TIM-3-expressing cell and contacting the APC with a cancerantigen.

BRIEF DESCRIPTION OF THE FIGURES

The following figures are provided for illustrative purposes only andare not required for understanding or practicing the invention.

FIG. 1A is a series of graphs depicting flow cytometric analyses ofvarious indicated cell types for expression of TIM-3. Th1, Th2, Tc1 andTc2 cells were stained with rat monoclonal antibody (mAb) to TIM-3(solid line) or isotype control (dotted line).

FIG. 1B is a graphical representation and comparison of deduced aminoacid sequences of murine TIM-3 (mTIM-3; SEQ ID NO:2) and human TIM-3(hTIM-3; SEQ ID NO:4), pointing out the IgV-like domain, mucin domain,transmembrane region, and cytoplasmic region for each.

FIG. 1C is a pair of graphs depicting flow cytometric analyses ofChinese hamster ovary (CHO) cells transfected with either mTIM-3 cDNA(CHO mTIM-3) or vector alone (CHO mock). Stable puromycin-resistantcells were stained with mAb to TIM-3 (solid line) or isotype control(dotted line).

FIG. 1D is a bar graph depicting relative expression of TIM-3 RNA tocontrol glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA as measuredby reverse transcriptase-polymerase chain reaction (RT-PCR) analysis oftotal RNA from various indicated cell lines and cells purified from SJLmice.

FIG. 2 is a series of graphs depicting flow cytometric analyses of TIM-3expression on the surface of Th1 DO11.10 CD4+ T cells (solid line,specific staining; dotted line, isotype control), plus intracellularexpression of IFN-γ, IL-4, and IL-10, after the various indicatednumbers of rounds of stimulation under Th1- and Th2-polarizingconditions.

FIG. 3A is a pair of bar graphs depicting relative expression of TIM-3RNA to control GAPDH RNA over time as measured by RT-PCR analysis oftotal RNA from lymph node (LN) and brain cells harvested from SJL miceat the indicated number of days following immunization with peptide PLP139-151 for the induction of EAE. Corresponding clinical disease scoresare as indicated.

FIG. 3B is a series of graphs depicting flow cytometric analyses ofTIM-3 expression on different indicated cell populations from brain,lymph node (LN), and spleen of SJL mice (solid line, specific staining;dotted line, isotype control) on day 10 following immunization with PLP139-151 for the induction of EAE.

FIGS. 4A-4F are a series of photomicrographs depicting the effects ofanti-TIM-3 antibody treatment on EAE. FIGS. 4A and 4B showinflammatory/demyelinating lesions in the spinal cord of an anti-TIM-3treated mouse on day 12 post-immunization at the peak of clinicaldisease. The infiltrate consists of a mixture of neutrophils andmononuclear cells. Perivascular fibrin deposition (arrow) indicatesvasculitis and vascular injury. A portion of an intact peripheral nerveroot (dark staining in FIG. 4B) is on the right side whereas most of thecentral nervous system (CNS) myelin in the field is lost. Mag.=411×.FIGS. 4C and 4D show extensive demyelination associated with aperivascular mononuclear cell infiltrate in the spinal cord posteriorcolumns of an anti-TIM-3-treated mouse sacrificed on day 30post-transfer. Inset (FIG. 4D): sheets of macrophages with phagocytosedmyelin fragments (dark dots). Mag.=137×; inset=411×. FIGS. 4E and 4Fshow a similar infiltrate in the posterior columns of an isotype controlmAb-treated mouse killed on day 30 post-immunization, with fewermacrophages and larger areas of intact (dark-staining) myelin.Mag.=137×. FIGS. 4A, 4C, and 4E, hematoxylin and eosin staining; FIGS.4B, 4D, and 4F, Klüver-Barrera staining for myelin.

FIG. 5A is a pair of graphs depicting in vitro proliferation ofsplenocytes taken from SJL mice 10 days after they were immunized withPLP 139-151 and given anti-TIM-3 or control antibody (rIgG), in responseto various indicated amounts of PLP 139-151 (PLP) or neuraminidase101-120 (Nase) peptide.

FIG. 5B is a series of graphs depicting flow cytometric analyses ofvarious indicated populations of splenocytes taken from SJL mice 10 daysafter they were immunized with PLP 139-151 and given anti-TIM-3 orcontrol antibody (rIgG).

FIG. 5C is a bar graph depicting proliferative response of indicatedpopulations of mixed and purified splenocytes taken from SJL mice 10days after they were immunized with PLP 139-151 and given anti-TIM-3 orcontrol antibody (rIgG2a). Gray bars, purified T cells and non-T cellsseparated by a permeable 0.2 μm membrane; black bars, no membrane.

FIG. 5D is a series of graphs depicting flow cytometric analyses ofspleen cells taken on day 3 from SJL mice injected on day 0 with 5×10⁶TIM-3-expressing, PLP 139-151 specific Th1 5B6 cells and then immunizedwith PLP 139-151. The recipients were also injected with anti-TIM-3 oranti-ICOS (control) antibody on days 0 and 2. FSC, forward scatter; SSC,side scatter.

FIG. 6A is a series of graphs depicting flow cytometric analyses ofCFSE-labeled T cells taken on day 3 from spleens and brains of SJL miceinjected on day 0 with 1×10⁷ TIM-3-expressing, PLP 139-151 specific Th15B6 cells and then immunized with PLP 139-151. The recipients were alsoinjected with anti-TIM-3, anti-ICOS (control), or anti-ICOSL (control)antibody on days 0 and 2.

FIG. 6B is a series of graphs depicting flow cytometric analyses ofCFSE-labeled T cells taken on day 7 from spleens and brains of SJL miceinjected on day 0 with 1×10⁷ TIM-3-expressing, PLP 139-151 specific Th15B6 cells and then immunized with PLP 139-151. The recipients were alsoinjected with anti-TIM-3, anti-ICOS (control), or anti-ICOSL antibody ondays 0, 2, 4, and 5.

FIG. 7 is a series of graphs depicting flow cytometric analyses ofvarious indicated cell lines (dendritic cell (DC), macrophage, and Bcell) as stained by a biotinylated soluble TIM-3 protein(TIM-3Ig-biotin).

FIG. 8A is a graph depicting proliferation of splenocytes obtained fromSJL mice immunized with PLP 139-151 peptide and treated for ten dayswith PBS, control hIgG, or soluble TIM-3 fusion protein (mTIM-3Ig/hFc ormTIM-3/hFc).

FIG. 8B is a pair of graphs depicting the stimulatory effect ofmTIM-3/hFc on secretion of Th1 cytokines IL-2 and IFN-γ.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to the novel appreciation of functionalcharacteristics of a molecule that is selectively expressed on thesurface of activated Th1 cells. The primary nucleotide and amino acidsequences of the particular molecule, here termed TIM-3, have previouslybeen described in U.S. Pat. No. 6,084,083. It has been discoveredaccording to the present invention that TIM-3 unexpectedly plays animportant role in the activation and proliferation of macrophages. Ithas also been surprisingly discovered according to the present inventionthat TIM-3-dependent activation and expansion of macrophages involves acognate interaction between the T cell expressing TIM-3 and themacrophage. Consistent with this finding, it has further been discoveredaccording to the present invention that antigen-presenting cells (APCs),including macrophages and dendritic cells, express a ligand or receptorfor TIM-3.

It has been discovered according to the present invention that moleculesthat bind to TIM-3, such as antibodies directed to TIM-3, can,surprisingly, induce macrophage activation and proliferation. Themacrophage activation induced by binding molecules directed againstTIM-3 promotes migration of the macrophages into target tissues,including tissue within the central nervous system (CNS). For example,it has been discovered according to the present invention that treatmentwith anti-TIM-3 of mice that are genetically susceptible of developingexperimental allergic encephalomyelitis (EAE), an in vivo model formultiple sclerosis, a Th1-type autoimmune disease of the brain inhumans, results in an unusually severe form of EAE characterized bymassive infiltration of activated macrophages into the CNS. Thusadministration of antibodies directed against TIM-3 unexpectedlyexacerbated, rather than ameliorated, this autoimmune disease.

Accordingly, in some aspects the present invention provides methods thatare useful for promoting APC activation, e.g., in treating or preventingintracellular infection, cancer, and autoimmune disease.

It has further been surprisingly discovered according to the presentinvention that TIM-3 expression on effector Th1 cells promotestrafficking of Th1 cells to target tissues. Unexpectedly,TIM-3-dependent trafficking of Th1 cells to target tissue can beaugmented with antibody for TIM-3. Thus in some aspects the presentinvention provides methods that are useful for promoting T-celltrafficking into a target tissue, e.g., in subjects in need of anenhanced immune response in the target tissue.

Furthermore, and importantly, it has surprisingly been discoveredaccording to the instant invention that TIM-3-dependent trafficking ofTh1 cells to target tissue can be inhibited with soluble TIM-3. Thus insome other aspects the present invention provides methods that areuseful for reducing T-cell trafficking into a target tissue, e.g., insubjects with autoimmune disease.

As used herein, “TIM-3” refers to the gene product encoded by thenucleotide sequence of SEQ ID NO:1 (murine), SEQ ID NO:3 (human), aswell as homologs, alleles, and functional variants thereof, e.g., SEQ IDNO:5. The gene product corresponding to the murine nucleotide sequenceof SEQ ID NO:1 is SEQ ID NO:2. The gene product corresponding to thehuman nucleotide sequence of SEQ ID NO:3 is SEQ ID NO:4; the geneproduct corresponding to the human nucleotide sequence of SEQ ID NO:5 isSEQ ID NO:6. The nucleotide sequence of SEQ ID NO:3 differs from that ofindependently determined SEQ ID NO:5 at one base, 476 (T in SEQ ID NO:3and G in SEQ ID NO:5). This single nucleotide difference results in achange of encoded amino acid from L (leucine) in SEQ ID NO:4 to R(arginine) in SEQ ID NO:6 at position 140. As shown in FIG. 1B, TIM-3 isa transmembrane protein that includes an extracellular region (includinga signal peptide, an IgV domain, and a mucin domain, each as describedfurther below), a transmembrane region, and a cytoplasmic region.Normally TIM-3 is preferentially expressed on the surface of activatedTh1 cells. TIM-3 cDNA sequences of the instant invention have beendeposited at the GenBank database under accession numbersAF450241-AF450243, shown below as SEQ ID NOs:1, 3, and 5, respectively.TIM-3 amino acid sequences of the instant invention have been depositedat the GenBank database under accession numbers AAL65156-AAL65158, shownbelow as SEQ ID NOs:2, 4, and 6, respectively.

Nucleotide sequence of murine TIM-3 cDNA SEQ ID NO: 1GenBank Accession No. AF450241ttttaaccga ggagctaaag ctatccctac acagagctgt ccttggattt cccctgccaa 60gtactcatgt tttcaggtct taccctcaac tgtgtcctgc tgctgctgca actactactt 120gcaaggtcat tggaagatgg ttataaggtt gaggttggta aaaatgccta tctgccctgc 180agttacactc tacctacatc tgggacactt gtgcctatgt gctggggcaa gggattctgt 240ccttggtcac agtgtaccaa tgagttgctc agaactgatg aaagaaatgt gacatatcag 300aaatccagca gataccagct aaagggcgat ctcaacaaag gagatgtgtc tctgatcata 360aagaatgtga ctctggatga ccatgggacc tactgctgca ggatacagtt ccctggtctt 420atgaatgata aaaaattaga actgaaatta gacatcaaag cagccaaggt cactccagct 480cagactgccc atggggactc tactacagct tctccaagaa ccctaaccac ggagagaaat 540ggttcagaga cacagacact ggtgaccctc cataataaca atggaacaaa aatttccaca 600tgggctgatg aaattaagga ctctggagaa acgatcagaa ctgctatcca cattggagtg 660ggagtctctg ctgggttgac cctggcactt atcattggtg tcttaatcct taaatggtat 720tcctgtaaga aaaagaagtt atcgagtttg agccttatta cactggccaa cttgcctcca 780ggagggttgg caaatgcagg agcagtcagg attcgctctg aggaaaatat ctacaccatc 840gaggagaacg tatatgaagt ggagaattca aatgagtact actgctacgt caacagccag 900cagccatcct gaccgcctct ggactgccac ttttaaaggc tcgccttcat ttctgacttt 960ggtatttccc tttttgaaaa ctatgtgata tgtcacttgg caacctcatt ggaggttctg 1020accacagcca ctgagaaaag agttccagtt ttctggggat aattaactca caaggggatt 1080cgactgtaac tcatgctaca ttgaaatgct ccattttatc cctgagtttc agggatcgga 1140tctcccactc cagagacttc aatcatgcgt gttgaagctc actcgtgctt tcatacatta 1200ggaatggtta gtgtgatgtc tttgagacat agaggtttgt ggtatatccg caaagctcct 1260gaacaggtag ggggaataaa gggctaagat aggaaggtgc ggttctttgt tgatgttgaa 1320aatctaaaga agttggtagc ttttctagag atttctgacc ttgaaagatt aagaaaaagc 1380caggtggcat atgcttaaca cgatataact tgggaacctt aggcaggagg gtgataagtt 1440caaggtcagc cagggctatg ctggtaagac tgtctcaaaa tccaaagacg aaaataaaca 1500tagagacagc aggaggctgg agatgaggct cggacagtga ggtgcatttt gtacaagcac 1560gaggaatcta tatttgatcg tagaccccac atgaaaaagc taggcctggt agagcatgct 1620tgtagactca agagatggag aggtaaaggc acaacagatc cccggggctt gcgtgcagtc 1680agcttagcct aggtgctgag ttccaagtcc acaagagtcc ctgtctcaaa gtaagatgga 1740ctgagtatct ggcgaatgtc catgggggtt gtcctctgct ctcagaagag acatgcacat 1800gaacctgcac acacacacac acacacacac acacacacac acacacacac acacatgaaa 1860tgaaggttct ctctgtgcct gctacctctc tataacatgt atctctacag gactctcctc 1920tgcctctgtt aagacatgag tgggagcatg gcagagcagt ccagtaatta attccagcac 1980tcagaaggct ggagcagaag cgtggagagt tcaggagcac tgtgcccaac actgccagac 2040tcttcttaca caagaaaaag gttacccgca agcagcctgc tgtctgtaaa aggaaaccct 2100gcgaaaggca aactttgact gttgtgtgct caaggggaac tgactcagac aacttctcca 2160ttcctggagg aaactggagc tgtttctgac agaagaacaa ccggtgactg ggacatacga 2220aggcagagct cttgcagcaa tctatatagt cagcaaaata ttctttggga ggacagtcgt 2280caccaaattg atttccaagc cggtggacct cagtttcatc tggcttacag ctgcctgccc 2340agtgcccttg atctgtgctg gctcccatct ataacagaat caaattaaat agaccccgag 2400tgaaaatatt aagtgagcag aaaggtagct ttgttcaaag atttttttgc attggggagc 2460aactgtgtac atcagaggac atctgttagt gaggacacca aaacctgtgg taccgttttt 2520tcatgtatga attttgttgt ttaggttgct tctagctagc tgtggaggtc ctggctttct 2580taggtgggta tggaagggag accatctaac aaaatccatt agagataaca gctctcatgc 2640agaagggaaa actaatctca aatgttttaa agtaataaaa ctgtactggc aaagtacttt 2700gagcatattt aaaaaaaaaa aaaaa 2725Nucleotide sequence of human TIM-3 cDNA, clone 1 SEQ ID NO: 3GenBank Accession No. AF450242ggagagttaa aactgtgcct aacagaggtg tcctctgact tttcttctgc aagctccatg 60ttttcacatc ttccctttga ctgtgtcctg ctgctgctgc tgctactact tacaaggtcc 120tcagaagtgg aatacagagc ggaggtcggt cagaatgcct atctgccctg cttctacacc 180ccagccgccc cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt 240gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg gacatccaga 300tactggctaa atggggattt ccgcaaagga gatgtgtccc tgaccataga gaatgtgact 360ctagcagaca gtgggatcta ctgctgccgg atccaaatcc caggcataat gaatgatgaa 420aaatttaacc tgaagttggt catcaaacca gccaaggtca cccctgcacc gactctgcag 480agagacttca ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag 540acacagacac tggggagcct ccctgatata aatctaacac aaatatccac attggccaat 600gagttacggg actctagatt ggccaatgac ttacgggact ctggagcaac catcagaata 660ggcatctaca tcggagcagg gatctgtgct gggctggctc tggctcttat cttcggcgct 720ttaattttca aatggtattc tcatagcaaa gagaagatac agaatttaag cctcatctct 780ttggccaacc tccctccctc aggattggca aatgcagtag cagagggaat tcgctcagaa 840gaaaacatct ataccattga agagaacgta tatgaagtgg aggagcccaa tgagtattat 900tgctatgtca gcagcaggca gcaaccctca caacctttgg gttgtcgctt tgcaatgcca 960tagatccaac caccttattt ttgagcttgg tgttttgtct ttttcagaaa ctatgagctg 1020tgtcacctga ctggttttgg aggttctgtc cactgctatg gagcagagtt ttcccatttt 1080cagaagataa tgactcacat gggaattgaa ctggga 1116Nucleotide sequence of human TIM-3 cDNA, clone 2 SEQ ID NO: 5GenBank Accession No. AF450243ggagagttaa aactgtgcct aacagaggtg tcctctgact tttcttctgc aagctccatg 60ttttcacatc ttccctttga ctgtgtcctg ctgctgctgc tgctactact tacaaggtcc 120tcagaagtgg aatacagagc ggaggtcggt cagaatgcct atctgccctg cttctacacc 180ccagccgccc cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt 240gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg gacatccaga 300tactggctaa atggggattt ccgcaaagga gatgtgtccc tgaccataga gaatgtgact 360ctagcagaca gtgggatcta ctgctgccgg atccaaatcc caggcataat gaatgatgaa 420aaatttaacc tgaagttggt catcaaacca gccaaggtca cccctgcacc gactctgcag 480agagacttca ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag 540acacagacac tggggagcct ccctgatata aatctaacac aaatatccac attggccaat 600gagttacggg actctagatt ggccaatgac ttacgggact ctggagcaac catcagaata 660ggcatctaca tcggagcagg gatctgtgct gggctggctc tggctcttat cttcggcgct 720ttaattttca aatggtattc tcatagcaaa gagaagatac agaatttaag cctcatctct 780ttggccaacc tccctccctc aggattggca aatgcagtag cagagggaat tcgctcagaa 840gaaaacatct ataccattga agagaacgta tatgaagtgg aggagcccaa tgagtattat 900tgctatgtca gcagcaggca gcaaccctca caacctttgg gttgtcgctt tgcaatgcca 960tagatccaac caccttattt ttgagcttgg tgttttgtct ttttcagaaa ctatgagctg 1020tgtcacctga ctggttttgg aggttctgtc cactgctatg gagcagagtt ttcccatttt 1080cagaagataa tgactcacat gggaattgaa ctggga 1116Amino acid sequence of murine TIM-3 SEQ ID NO: 2GenBank Accession No. AAL65156MFSGLTLNCV LLLLQLLLAR SLEDGYKVEV GKNAYLPCSY TLPTSGTLVP MCWGKGFCPW 60SQCTNELLRT DERNVTYQKS SRYQLKGDLN KGDVSLIIKN VTLDDHGTYC CRIQFPGLMN 120DKKLELKLDI KAAKVTPAQT AHGDSTTASP RTLTTERNGS ETQTLVTLHN NNGTKISTWA 180DEIKDSGETI RTAIHIGVGV SAGLTLALII GVLILKWYSC KKKKLSSLSL ITLANLPPGG 240LANAGAVRIR SEENIYTIEE NVYEVENSNE YYCYVNSQQP S 281Amino acid sequence of human TIM-3, clone 1 SEQ ID NO: 4GenBank Accession No. AAL65157MFSHLPFDCV LLLLLLLLTR SSEVEYRAEV GQNAYLPCFY TPAAPGNLVP VCWGKGACPV 60FECGNVVLRT DERDVNYWTS RYWLNGDFRK GDVSLTIENV TLADSGIYCC RIQIPGIMND 120EKFNLKLVIK PAKVTPAPTL QRDFTAAFPR MLTTRGHGPA ETQTLGSLPD INLTQISTLA 180NELRDSRLAN DLRDSGATIR IGIYIGAGIC AGLALALIFG ALIFKWYSHS KEKIQNLSLI 240SLANLPPSGL ANAVAEGIRS EENIYTIEEN VYEVEEPNEY YCYVSSRQQP SQPLGCRFAM 300 P301 Amino acid sequence of human TIM-3, clone 2 SEQ ID NO: 6GenBank Accession No. AAL65158MFSHLPFDCV LLLLLLLLTR SSEVEYRAEV GQNAYLPCFY TPAAPGNLVP VCWGKGACPV 60FECGNVVLRT DERDVNYWTS RYWLNGDFRK GDVSLTIENV TLADSGIYCC RIQIPGIMND 120EKFNLKLVIK PAKVTPAPTR QRDFTAAFPR MLTTRGHGPA ETQTLGSLPD INLTQISTLA 180NELRDSRLAN DLRDSGATIR IGIYIGAGIC AGLALALIFG ALIFKWYSHS KEKIQNLSLI 240SLANLPPSGL ANAVAEGIRS EENIYTIEEN VYEVEEPNEY YCYVSSRQQP SQPLGCRFAM 300 P301

Functional variants of TIM-3 include molecules representing mutations,additions, deletions, and truncations of full-length TIM-3, providedsuch molecules retain at least one functional characteristic offull-length TIM-3. For example, a TIM-3 molecule truncated so as to lackmost or all of its cytoplasmic domain is expected to retain the abilityto bind to ligands or receptors for TIM-3.

Certain aspects of the invention involve methods which promote T-celltrafficking into selected tissues.

In one aspect the invention provides a method for treating a subject inneed of an enhanced immune response in a target tissue. The methodinvolves administering to the subject a TIM-3-binding molecule in aneffective amount to promote T-cell trafficking to the target tissue.

In another aspect the invention provides a method for treating a subjectin need of treatment for a tumor. The method according to this aspect ofthe invention involves administering to a subject in need of treatmentfor a tumor a TIM-3-binding molecule in an effective amount to promoteT-cell trafficking to the tumor.

In yet another aspect the invention provides a method for treating asubject in need of treatment for an infection. The method according tothis aspect of the invention involves administering to a subject in needof treatment for an infection a TIM-3-binding molecule in an effectiveamount to promote T-cell trafficking to the infection.

As used herein, “treat” and “treating” refer to a therapeuticintervention in a subject to prevent the onset of, alleviate thesymptoms of, or slow or stop the progression of a disorder or diseasebeing treated in the subject. The therapeutic intervention can be theadministration of a therapeutically effective amount of a substance toprevent the onset of, alleviate the symptoms of, or slow or stop theprogression of a disorder or disease being treated.

A “subject” as used herein refers to any vertebrate animal, preferably amammal, and more preferably a human. Examples of subjects includehumans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs,goats, cows, horses, dogs, cats, birds, and fish.

A “subject in need of an enhanced immune response” as used herein refersto a subject having or at risk of having a disease, disorder, orcondition that is associated with a deficient or absent immune response,or that can be relieved by augmenting an immune response. Subjects inneed of an enhanced immune response are common and are readilyrecognized by those of skill in the art. The very young, the elderly,subjects with chronic disease or acute-on-chronic disease, subjects withsusceptibility to infection due to compromised barriers to infection(including subjects with cystic fibrosis), subjects with drug-inducedimmune deficiency, critically ill subjects, subjects about to undergosurgery, subjects with congenital or genetic forms of immunodeficiency,subjects with acquired forms of immunodeficiency (including subjectsinfected with human immunodeficiency virus, HIV), subjects withpersistent infection, subjects with intracellular infection, andsubjects with cancer are all subjects in need of an enhanced immuneresponse. This list is meant to be representative and not limiting inany way.

A “target tissue” as used herein refers to a tissue representing a siteof immune effector activity. A target tissue can be any tissue in asubject. Examples of target tissues include brain or central nervoussystem, breast, lung, kidney, liver, pancreas (including in particularpancreatic islets), stomach, intestine, ovary, uterus, testis, prostate,marrow, bone, joint synovia, muscle, and skin. Typically, target tissuesare tissues not primarily associated with lymphoid tissues, except insituations involving cancers, infections, and inflammatory conditions ofthose tissues. Thus target tissues can, but typically do not, includelymph nodes, spleen, mucosal lymphoid tissues (including, e.g., Peyer'spatches), or thymus.

As used herein, a “TIM-3-binding molecule” is any molecule that bindsspecifically to TIM-3. The TIM-3-binding molecule can be a smallmolecule, a polypeptide, an antibody or a fragment of an antibody, apolynucleotide, a carbohydrate including a polysaccharide, a lipid, adrug, as well as mimics, derivatives, and combinations thereof. TheTIM-3-binding molecule can be found in nature or it can be derived orsynthesized using suitable in vitro and synthetic methods known by thoseof skill in the art. For example, the TIM-3-binding molecule can be asmall molecule that is identified through screening a library of smallmolecules for the ability to bind to TIM-3.

The TIM-3-binding molecule can be generated and identified using phagedisplay of peptides. As yet another example, the TIM-3-binding moleculecan be a TIM-3 ligand, including a soluble TIM-3 ligand. A “TIM-3ligand” as used herein refers to a type of TIM-3-binding molecule thatbinds specifically to the extracellular region of TIM-3. TIM-3 ligand isa naturally occurring receptor or counter-receptor for TIM-3, and it isbelieved to be expressed on certain cells of the immune system,including macrophages and dendritic cells. It is also believed thatTIM-3 ligand can also be expressed on certain other cells that come incontact with TIM-3 expressed on T cells, e.g., endothelial cells,mucosal epithelial cells, and the like. Engagement of TIM-3 ligand byTIM-3 can deliver a signal to the interior of the cell expressing TIM-3ligand on its surface and/or the cell expressing TIM-3 on its surface. ATIM-3 ligand also refers to any TIM-3-binding molecule that competeswith a naturally occurring TIM-3 ligand for binding to TIM-3. A TIM-3ligand thus includes but is not limited to a naturally occurring TIM-3ligand that binds to TIM-3.

“Soluble TIM-3 ligand” refers to any form of TIM-3 ligand that isdissociated from cell membrane. Soluble TIM-3 ligand can be a C-terminaltruncated form of full-length TIM-3 ligand or a transmembrane-deletedversion of TIM-3 ligand. In one embodiment soluble TIM-3 ligand refersto a fusion protein that includes at least an extracellular domain ofTIM-3 ligand and another polypeptide. In one embodiment the solubleTIM-3 ligand is a fusion protein including the extracellular region ofTIM-3 ligand covalently linked, e.g., via a peptide bond, to an Fcfragment of an immunoglobulin such as IgG.

In some embodiments the TIM-3-binding molecule is an antibody specificfor TIM-3 or is a fragment of an antibody specific for TIM-3. An“antibody specific for TIM-3” as used herein refers to an immunoglobulinthat binds specifically to a TIM-3 epitope through interaction betweenthe epitope and a variable domain of the immunoglobulin. A “fragment ofan antibody specific for TIM-3” shall refer to a portion of an intactantibody specific for TIM-3 that binds specifically to a TIM-3 epitopethrough interaction between the epitope and a variable domain of theintact immunoglobulin from which the fragment is derived. A “fragment ofan antibody specific for TIM-3” shall also refer to an engineeredequivalent of a portion of an intact antibody specific for TIM-3 thatbinds specifically to a TIM-3 epitope through interaction between theepitope and a variable domain. As is well known in the art, intactantibodies generally include both variable domains and at least oneconstant domain. The variable domain includes contributions from heavyand light chains that together provide stretches of contact residuesspecific for the binding of the antibody with an antigen. The constantdomain is not specific for the antigen but rather is more or less commonto all antibodies of a particular isotype; it may be involved in bindingcomplement or antigen-independent binding of the antibody to Fcreceptors expressed on certain immune effector cells. The antibody canbe monoclonal or polyclonal. Furthermore, the antibody can be native orit can be engineered in part or in whole to reduce its potentialimmunogenicity in a treated host. Methods for generating and isolatingpolyclonal and monoclonal antibodies specific for a given antigen arewell described in the art. See, for example, Kohler and Milstein (1975)Nature 256:495-7; Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y., current edition.

The antibody specific for TIM-3 is meant to encompass antibodies derivedfrom any appropriate species. For example, antibodies for a particularantigen can be raised by immunizing an appropriate host with theantigen. The immunized host can be selected from a variety of species,including, for example, mouse, rat, hamster, guinea pig, rabbit, goat,sheep, horse, monkey, and human. Methods for generating chimeric andhumanized monoclonal antibodies are also well known in the art, andexamples of such antibodies are in clinical use today. In addition tothe species of origin of the antibody, those of skill in the artrecognize that various isotypes or classes of immunoglobulin exist.These include, for example, IgG, IgA, IgE, IgM, and IgD. Within theseclasses there can also be further subclasses or subtypes, e.g., humanIgG subtypes include IgG1, IgG2, IgG3, and IgG4, while murine IgGsubtypes include IgG1, IgG2a, IgG2b, and IgG3. The antibody specific forTIM-3 is meant to encompass any isotype and subtype. In some embodimentsthe antibody is an IgG.

In some embodiments the TIM-3-binding molecule binds to an extracellularregion of TIM-3. The term “extracellular region of TIM-3” refers to thatportion of the expressed TIM-3 gene product that normally residessubstantially on the extracellular surface of a cell expressing theTIM-3 gene product. In accordance with the present invention, thepredicted amino acid sequence of TIM-3 includes an extracellular region,a transmembrane region, and a cytoplasmic or intracellular region. Theextracellular region is predicted to include the N-terminal 191 aminoacid residues of murine TIM-3 and the N-terminal 200 amino acid residuesof human TIM-3, inclusive of a 21 amino acid signal peptide in eachinstance. The signal peptide can be cleaved from the expressed proteinproduct so that the extracellular region of murine TIM-3 is 170 aminoacids long and the extracellular region of human TIM-3 is 179 aminoacids long.

The extracellular region of TIM-3 is believed to include at least twodomains, an IgV domain and a mucin domain (see FIG. 1B). The IgV domainof TIM-3 shares structural similarities with an immunoglobulin variabledomain, and it is believed to occupy amino acids 22-132 in murine TIM-3and amino acids 22-131 in human TIM-3. The mucin domain is believed tooccupy amino acids 133-191 in murine TIM-3 and amino acids 132-200 inhuman TIM-3.

As used herein, the expression “IgV domain or a fragment thereof” refersto the full-length IgV domain of the extracellular region of TIM-3 or toa portion of the full-length IgV domain of the extracellular region ofTIM-3 sufficiently long to be used as an antigen for immunization of ahost. It is generally believed that a linear determinant of a proteinantigen that forms contacts with a specific antibody is about six aminoacids long. Thus typically the fragment will include at least sixcontiguous amino acids according to SEQ ID NO:2 or SEQ ID NO:4 includedin the IgV domain as specified above.

As used herein, the term “mucin domain or a fragment thereof” refers tothe full-length mucin domain of the extracellular region of TIM-3 or toa portion of the full-length mucin domain of the extracellular region ofTIM-3 sufficiently long to be used as an antigen for immunization of ahost. A fragment will typically include at least six contiguous aminoacids according to SEQ ID NO:2 or SEQ ID NO:4 included in the mucindomain as specified above.

An “effective amount” as used herein is any amount that is sufficienteither to promote the occurrence of a desired outcome or condition, orto reduce or inhibit the occurrence of an undesired outcome orcondition. In some instances a desired outcome or condition is an idealthat represents one end of a spectrum of possible outcomes orconditions. In such instances an effective amount is any amountassociated with an outcome or condition that is closer to the desiredideal than would be achieved or observed without the effective amount.Thus an effective amount promotes the occurrence of a desired outcome orcondition, but it need not achieve an ultimate endpoint.

As used herein, “T-cell trafficking” refers to migration of Tlymphocytes to a site of immune response activity. Naïve T cellsrecirculate throughout the body, leaving and reentering the lymphoidtissues as they sample their environment for the presence of non-selfantigens or “danger” signals. Lymphoid tissues are specially adapted tohelp promote encounters between antigen-specific T-cell receptorsexpressed on T cells and their cognate antigens. Specializedantigen-presenting cells (APCs) concentrate within lymphoid tissues, andare specially adapted to interact with and to present antigens to Tcells to initiate an immune response by T cells genetically programmedto recognize a particular antigen. Following T-cell activation inresponse to encounter with specific antigen, T cells proliferate,undergo differentiation to produce a variety of secreted andcell-associated products, including cytokines, and migrate to tissuesites associated with the antigen. The result of this process is thatnaïve T cells circulate randomly while activated T cells proliferate andhome to specific tissue sites.

In some embodiments the subject has cancer or is at risk of havingcancer. A “cancer” as used herein refers to an uncontrolled growth ofcells which interferes with the normal functioning of the bodily organsand systems. A subject that has a cancer is a subject having objectivelymeasurable cancer cells present in the subject's body. A subject at riskof having a cancer is a subject that is predisposed to develop a cancer.Such a subject can include, for example, a subject with a family historyof or a genetic predisposition toward developing a cancer. A subject atrisk of having a cancer also can include a subject with a known orsuspected exposure to a cancer-causing agent.

Cancers which migrate from their original location and seed vital organscan eventually lead to the death of the subject through the functionaldeterioration of the affected organs. Hemopoietic cancers, such asleukemia, are able to out-compete the normal hemopoietic compartments ina subject, thereby leading to hemopoietic failure (in the form ofanemia, thrombocytopenia and neutropenia) ultimately causing death.

A metastasis is a region of cancer cells, distinct from the primarytumor location resulting from the dissemination of cancer cells from theprimary tumor to other parts of the body. At the time of diagnosis ofthe primary tumor mass, the subject may be monitored for the presence ofmetastases. Metastases are most often detected through the sole orcombined use of magnetic resonance imaging (MRI) scans, computedtomography (CT) scans, blood and platelet counts, liver functionstudies, chest X-rays and bone scans in addition to the monitoring ofspecific symptoms.

Cancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer; cervical cancer; choriocarcinoma; colon and rectum cancer;connective tissue cancer; cancer of the digestive system; endometrialcancer; esophageal cancer; eye cancer; cancer of the head and neck;gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small cell and non-smallcell); lymphoma including Hodgkin's and non-Hodgkin's lymphoma;melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; thyroid cancer; uterine cancer; cancer of the urinary system, aswell as other carcinomas and sarcomas.

In some preferred embodiments the subject has an infection or is at riskof having an infection. An “infection” as used herein refers to adisease or condition attributable to the presence in a host of a foreignorganism or agent that reproduces within the host. Infections typicallyinvolve breach of a normal mucosal or other tissue barrier by aninfectious organism or agent. A subject that has an infection is asubject having objectively measurable infectious organisms or agentspresent in the subject's body. A subject at risk of having an infectionis a subject that is predisposed to develop an infection. Such a subjectcan include, for example, a subject with a known or suspected exposureto an infectious organism or agent. A subject at risk of having aninfection also can include a subject with a condition associated withimpaired ability to mount an immune response to an infectious organismor agent, e.g., a subject with a congenital or acquiredimmunodeficiency, a subject undergoing radiation therapy orchemotherapy, a subject with a burn injury, a subject with a traumaticinjury, a subject undergoing surgery or other invasive medical or dentalprocedure.

Infections are broadly classified as bacterial, viral, fungal, orparasitic based on the category of infectious organism or agentinvolved. Other less common types of infection are also known in theart, including, e.g., infections involving rickettsiae, mycoplasmas, andagents causing scrapie, bovine spongiform encephalopthy (BSE), and priondiseases (e.g., kuru and Creutzfeldt-Jacob disease). Examples ofbacteria, viruses, fungi, and parasites which cause infection are wellknown in the art. An infection can be acute, subacute, chronic, orlatent, and it can be localized or systemic. Furthermore, an infectioncan be predominantly intracellular or extracellular during at least onephase of the infectious organism's or agent's life cycle in the host.

Bacteria include both Gram negative and Gram positive bacteria. Examplesof Gram positive bacteria include, but are not limited to Pasteurellaspecies, Staphylococci species, and Streptococcus species. Examples ofGram negative bacteria include, but are not limited to, Escherichiacoli, Pseudomonas species, and Salmonella species. Specific examples ofinfectious bacteria include but are not limited to: Helicobacterpyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteriaspp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii,M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group AStreptococcus), Streptococcus agalactiae (Group B Streptococcus),Streptococcus (viridans group), Streptococcus faecalis, Streptococcusbovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae,pathogenic Campylobacter spp., Enterococcus spp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diphtheriae,Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides spp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelii.

Examples of virus that have been found to cause infections in humansinclude but are not limited to: Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III),HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviviridae(e.g., dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the etiological agents of Spongiform encephalopathies, the agentof delta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1=enterallytransmitted; class 2=parenterally transmitted (i.e., Hepatitis C);Norwalk and related viruses, and astroviruses).

Examples of fungi include: Aspergillus spp., Blastomyces dermatitidis,Candida albicans, other Candida spp., Coccidioides immitis, Cryptococcusneoformans, Histoplasma capsulatum, Chlamydia trachomatis, Nocardiaspp., Pneumocystis carinii.

Parasites include but are not limited to blood-borne and/or tissuesparasites such as Babesia microti, Babesia divergens, Entamoebahistolytica, Giardia lamblia, Leishmania tropica, Leishmania spp.,Leishmania braziliensis, Leishmania donovani, Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Toxoplasmagondii, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasmagondii, flat worms, round worms.

As used herein, “lymph node associated with the target tissue” refers toany lymph node or other lymphoid tissue to which lymph circulating froma tissue is expected or shown to return. As mentioned above, lymphocyteswithin the blood leave the circulation to sample tissues. In order toreturn to the circulation, lymphocytes in a tissue make their waythrough lymphatic endothelium into the lymphatic circulation, flowing toa draining lymph node via afferent lymphatics. The anatomy andassociation of lymph nodes and the tissues they serve are well known tothose of skill in the art. A given target tissue can have more than onedraining lymph node. Nonlimiting examples of lymph nodes include aortic,axillary, bronchopulmonary, buccal, celiac, cervical, cystic,deltopectoral, iliac, infraclavicular, inguinal, intercostal, internalthoracic, jugulodigastric, jugulo-omohyoid, lumbar, mastoid,mediastinal, mesenteric, occipital, para-aortic, pararectal, parotid,pectoral, popliteal, preaortic, pulmonary, retroauricular,retropharyngeal, submandibular, submental, subscapular, supratrochlear,tonsils, tracheobroncheal.

Also according to this aspect of the invention, in some embodiments themethod further entails administering to the subject an adjuvant. An“adjuvant” as used herein refers to an antigen-nonspecific stimulator ofthe immune response. The use of adjuvants is essential to induce astrong antibody response to soluble antigens (Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. CurrentEdition; hereby incorporated by reference). The overall effect ofadjuvants is dramatic and their importance cannot be overemphasized. Theaction of an adjuvant allows much smaller doses of antigen to be usedand generates antibody responses that are more persistent. Thenonspecific activation of the immune response often can spell thedifference between success and failure in obtaining an immune response.Adjuvants should be used for first injections unless there is some veryspecific reason to avoid this. Most adjuvants incorporate twocomponents. One component is designed to protect the antigen from rapidcatabolism (e.g., liposomes or synthetic surfactants (Hunter et al.1981)). Liposomes are only effective when the immunogen is incorporatedinto the outer lipid layer; entrapped molecules are not seen by theimmune system. The other component is a substance that will stimulatethe immune response nonspecifically. These substances act by raising thelevel of lymphokines Lymphokines stimulate the activity ofantigen-processing cells directly and cause a local inflammatoryreaction at the site of injection. Early work relied entirely onheat-killed bacteria (Dienes 1936) or lipopolysaccaride (LPS) (Johnsonet al. 1956). LPS is reasonably toxic, and, through analysis of itsstructural components, most of its properties as an adjuvant have beenshown to be in a portion known as lipid A. Lipid A is available in anumber of synthetic and natural forms that are much less toxic than LPSbut still retain most of the better adjuvant properties of parental LPSmolecule. Lipid A compounds are often delivered using liposomes.

Adjuvants include, but are not limited to, alum (e.g., aluminumhydroxide, aluminum phosphate); saponins purified from the bark of theQ. saponaria tree, such as QS21 (a glycolipid that elutes in the 21^(st)peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc.,Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA); derivatives of lipopolysaccharides suchas monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc.,Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonyl-muramyldipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related tolipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.), emulsion-based formulations including mineral oil, non-mineraloil, water-in-oil or oil-in-water-in oil emulsion, oil-in-wateremulsions such as Seppic ISA series of Montanide adjuvants; and PROVAX,ISCOMs (Immunostimulating complexes which contain mixed saponins, lipidsand form virus-sized particles with pores that can hold antigen; SB-AS2(SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsioncontaining MPL and QS21: SmithKline Beecham Biologicals [SBB],Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 whichcontains alum and MPL; SBB, Belgium); non-ionic block copolymers thatform micelles such as CRL 1005 (these contain a linear chain ofhydrophobic polyoxpropylene flanked by chains of polyoxyethylene;Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, anoil-in-water emulsion containing Tween 80 and a nonionic blockcopolymer; Syntex Chemicals, Inc., Boulder, Colo.).

In some embodiments the method according to this aspect of the inventionfurther involves administering to the subject an anti-tumor medicament.As used herein, an “anti-tumor medicament” or, equivalently, a “cancermedicament”, refers to an agent which is administered to a subject forthe purpose of treating a cancer. As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers. Additionally, the methods of theinvention are intended to embrace the use of more than one cancermedicament along with the TIM-3-binding molecule of the presentinvention. As an example, where appropriate, the TIM-3-binding moleculecan be administered with a both a chemotherapeutic agent and animmunotherapeutic agent. Alternatively, the cancer medicament canembrace an immunotherapeutic agent and a cancer vaccine, or achemotherapeutic agent and a cancer vaccine, or a chemotherapeuticagent, an immunotherapeutic agent and a cancer vaccine all administeredto one subject for the purpose of treating a subject having a cancer orat risk of developing a cancer.

Cancer medicaments function in a variety of ways. Some cancermedicaments work by targeting physiological mechanisms that are specificto tumor cells. Examples include the targeting of specific genes andtheir gene products (i.e., proteins primarily) which are mutated incancers. Such genes include but are not limited to oncogenes (e.g., Ras,Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cellcycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments canalternately target signal transduction pathways and molecular mechanismswhich are altered in cancer cells. Targeting of cancer cells via theepitopes expressed on their cell surface is accomplished through the useof monoclonal antibodies. This latter type of cancer medicament isgenerally referred to herein as immunotherapy.

Other cancer medicaments target cells other than cancer cells. Forexample, some medicaments prime the immune system to attack tumor cells(i.e., cancer vaccines). Still other medicaments, called angiogenesisinhibitors, function by attacking the blood supply of solid tumors.Since the most malignant cancers are able to metastasize (i.e., exit theprimary tumor site and seed a distal tissue, thereby forming a secondarytumor), medicaments that impede this metastasis are also useful in thetreatment of cancer. Angiogenesis inhibitors include basic FGF (b-FGF),VEGF, angiopoietins, angiostatin, endostatin, TNF-α, TNP-470,thrombospondin-1, platelet factor 4, CAI, and certain members of theintegrin family of proteins. One category of this type of medicament isa metalloproteinase inhibitor, which inhibits the enzymes used by thecancer cells to exist the primary tumor site and extravasate intoanother tissue.

As used herein, chemotherapeutic agents embrace all other forms ofcancer medicaments which do not fall into the categories ofimmunotherapeutic agents or cancer vaccines. Chemotherapeutic agents asused herein encompass both chemical and biological agents. These agentsfunction to inhibit a cellular activity upon which the cancer celldepends for continued survival. Categories of chemotherapeutic agentsinclude alkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most if not all ofthese agents are directly toxic to cancer cells and do not requireimmune stimulation.

Chemotherapeutic agents which are currently in development or in use ina clinical setting include, without limitation: 5-FU Enhancer, 9-AC,AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide, Amsacrine(m-AMSA), Angiogenesis Inhibitor, Anti-VEGF, Asparaginase, Azacitidine,Batimastat (BB94), BAY 12-9566, BCH-4556, Bis-Naphtalimide, Busulfan,Capecitabine, Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2inhibitors, Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682,Cytarabine HCl, D2163, Dactinomycin, Daunorubicin HCl, DepoCyt,Dexifosamide, Docetaxel, Dolastain, Doxifluridine, Doxorubicin, DX8951f,E 7070, EGFR, Epirubicin, Erythropoietin, Estramustine phosphate sodium,Etoposide (VP16-213), Farnesyl Transferase Inhibitor, FK 317,Flavopiridol, Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide,Fragyline, Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b,Interleukin-2, Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolideacetate (LHRH-releasing factor analogue), Levamisole, LiGLA(lithium-gamma linolenate), Lodine Seeds, Lometexol, Lomustine (CCNU),Marimistat, Mechlorethamine HCl (nitrogen mustard), Megestrol acetate,Meglamine GLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone,Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698,OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARPInhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412,Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS FamesylTransferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU),Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide,Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase,UFT (Tegafur/Uracil), Valrubicin, VEGF/b-FGF Inhibitors, Vinblastinesulfate, Vindesine sulfate, VX-710, VX-853, YM 116, ZD 0101, ZD0473/Anormed, ZD 1839, ZD 9331.

Immunotherapeutic agents are medicaments which derive from antibodies orantibody fragments which specifically bind or recognize a cancerantigen. The goal of immunotherapy is to augment a patient's immuneresponse to an established tumor. One method of immunotherapy includesthe use of adjuvants. Adjuvant substances derived from microorganisms,such as bacillus Calmette-Guérin, heighten the immune response andenhance resistance to tumors in animals.

Some cancer cells are antigenic and thus can be targeted by the immunesystem. In one aspect, the combined administration of TIM-3-bindingmolecule and cancer medicaments, particularly those which are classifiedas cancer immunotherapies, is useful for stimulating a specific immuneresponse against a tumor antigen.

As used herein, the terms “tumor antigen” and “cancer antigen” are usedinterchangeably to refer to antigens which are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

A tumor antigen is typically a peptide associated with the surface of atumor or cancer cell and which is capable of provoking an immuneresponse when expressed on the surface of an APC in the context of anMHC molecule. Cancer antigens, such as those present in cancer vaccinesor those used to prepare cancer immunotherapies, can be prepared fromcrude cancer cell extracts, as described in Cohen P A et al. (1994)Cancer Res 54:1055-8, or by partially purifying the antigens, usingrecombinant technology, or de novo synthesis of known antigens. Cancerantigens can be used in the form of immunogenic portions of a particularantigen or in some instances a whole cell or a tumor mass can be used asthe antigen. Such antigens can be isolated or prepared recombinantly orby any other means known in the art.

The theory of immune surveillance is that a prime function of the immunesystem is to detect and eliminate neoplastic cells before a tumor forms.A basic principle of this theory is that cancer cells are antigenicallydifferent from normal cells and thus elicit immune reactions that aresimilar to those that cause rejection of immunologically incompatibleallografts. Studies have confirmed that tumor cells differ, eitherqualitatively or quantitatively, in their expression of antigens. Forexample, “tumor-specific antigens” are antigens that are specificallyassociated with tumor cells but not normal cells. Examples oftumor-specific antigens are viral antigens in tumors induced by DNA orRNA viruses. “Tumor-associated” antigens are present in both tumor cellsand normal cells but are present in a different quantity or a differentform in tumor cells. Examples of such antigens are oncofetal antigens(e.g., carcinoembryonic antigen), differentiation antigens (e.g., T andTn antigens), and oncogene products (e.g., HER/neu).

As previously noted, the polypeptide products of tumor-specific genescan be the targets for host immune surveillance and provoke selectionand expansion of one or more clones of CTLs specific for thetumor-specific gene product. Examples of this phenomenon includeproteins and fragments thereof encoded by the MAGE family of genes. InPCT application PCT/US92/04354, published on Nov. 26, 1992, the “MAGE”family, a tumor-specific family of genes, is disclosed. The expressionproducts of these genes are processed into peptides which, in turn, areexpressed on cell surfaces. This can lead to lysis of the tumor cells byspecific CTLs. The genes are said to code for “tumor rejection antigenprecursors” or “TRAP” molecules, and the peptides derived therefrom arereferred to as “tumor rejection antigens” or “TRAs”. See Traversari C etal. (1992) Immunogenetics 35:145-52; van der Bruggen P et al. (1991)Science 254:1643-47, for further information on this family of genes.Also, see U.S. Pat. No. 5,342,774.

In addition to the MAGE family of genes, tumor antigens are also encodedby the MAGE-Xp family of genes (U.S. Pat. No. 5,587,289), the tyrosinasegene (PCT publication WO94/14459), the Melan-A gene (PCT publicationWO94/21126), the BAGE gene (U.S. Pat. No. 5,571,711 and PCT publicationWO95/00159), the GAGE gene (U.S. Pat. No. 5,610,013 and PCT publicationWO95/03422), the RAGE family of genes (U.S. Pat. No. 5,939,526), thePRAME (formerly DAGE) gene (PCT publication WO96/10577), theMUM-1/LB-33B gene (U.S. Pat. No. 5,589,334), the NAG gene (U.S. Pat. No.5,821,122), the FB5 (endosialin) gene (U.S. Pat. No. 6,217,868), and thePMSA gene (U.S. Pat. No. 5,939,818). The foregoing list is only intendedto be representative and is not to be understood to be limiting.

Different types of cells that can kill tumor targets in vitro and invivo have been identified: natural killer cells (NK cells), cytotoxic Tlymphocytes (CTLs), lymphokine-activated killer cells (LAKs), andactivated macrophages. NK cells can kill tumor cells without having beenpreviously sensitized to specific antigens, and the activity does notrequire the presence of class I antigens encoded by the majorhistocompatibility complex (MHC) on target cells. NK cells are thoughtto participate in the control of nascent tumors and in the control ofmetastatic growth. In contrast to NK cells, CTLs can kill tumor cellsonly after they have been sensitized to tumor antigens and when thetarget antigen is expressed on the tumor cells that also express MHCclass I. CTLs are thought to be effector cells in the rejection oftransplanted tumors and of tumors caused by DNA viruses. LAK cells are asubset of null lymphocytes distinct from the NK cell and CTLpopulations. Activated macrophages can kill tumor cells in a manner thatis not antigen-dependent nor MHC-restricted once activated. Activatedmacrophages are thought to decrease the growth rate of the tumors theyinfiltrate. In vitro assays have identified other immune mechanisms suchas antibody-dependent, cell-mediated cytotoxic reactions and lysis byantibody plus complement. However, these immune effector mechanisms arethought to be less important in vivo than the function of NK cells,CTLs, LAK cells, and macrophages in vivo (for review see Piessens W Fand David J, “Tumor Immunology”, In: Scientific American Medicine, Vol.2, Scientific American Books, N.Y., pp. 1-13, 1996).

In some embodiments the anti-tumor medicament includes a tumor-specificantibody or tumor-specific fragment thereof. The term “tumor-specificantibody” refers to an antibody that specifically binds to a tumorantigen. A “tumor-specific antibody fragment” as used herein refers to afragment of a tumor-specific antibody that binds specifically to a tumorantigen. Typically the fragment includes at least part of a variabledomain including a hypervariable region that contributes to antigenspecificity and binding. Examples of tumor-specific antibody fragmentsinclude, without limitation, Fab, Fv, Fab′, and F(ab′)₂ fragmentsderived from tumor-specific antibodies.

Preferably, the tumor antigen is expressed at the cell surface of thecancer cell. Even more preferably, the antigen is one which is notexpressed by normal cells, or at least not expressed to the same levelas in cancer cells. Antibody-based immunotherapies may function bybinding to the cell surface of a cancer cell and thereby stimulate theendogenous immune system to attack the cancer cell. Another way in whichantibody-based therapy functions is as a delivery system for thespecific targeting of toxic substances to cancer cells. Antibodies areusually conjugated to toxins such as ricin (e.g., from castor beans),calicheamicin and maytansinoids, to radioactive isotopes such asIodine-131 and Yttrium-90, to chemotherapeutic agents (as describedherein), or to biological response modifiers. In this way, the toxicsubstances can be concentrated in the region of the cancer andnon-specific toxicity to normal cells can be minimized.

In addition to the use of antibodies which are specific for cancerantigens, antibodies which bind to vasculature, such as those which bindto endothelial cells, are also useful in the invention. Solid tumorsgenerally are dependent upon newly formed blood vessels to survive, andthus most tumors are capable of recruiting and stimulating the growth ofnew blood vessels. As a result, one strategy of many cancer medicamentsis to attack the blood vessels feeding a tumor and/or the connectivetissues (or stroma) supporting such blood vessels.

Examples of cancer immunotherapies which are currently being used orwhich are in development include but are not limited to Rituxan,IDEC-C2B8, anti-CD20 Mab, Panorex, 3622W94, anti-EGP40 (17-1A)pancarcinoma antigen on adenocarcinomas Herceptin, anti-Her2, Anti-EGFr,BEC2, anti-idiotypic-GD₃ epitope, Ovarex, B43.13, anti-idiotypic CA125,4B5, Anti-VEGF, RhuMAb, MDX-210, anti-HER-2, MDX-22, MDX-220, MDX-447,MDX-260, anti-GD-2, Quadramet, CYT-424, IDEC-Y2B8, Oncolym, Lym-1, SMARTM195, ATRAGEN, LDP-03, anti-CAMPATH, ior t6, anti CD6, MDX-11, OV103,Zenapax, Anti-Tac, anti-IL-2 receptor, MELIMMUNE-2, MELIMMUNE-1,CEACIDE, Pretarget, NovoMAb-G2, TNT, anti-histone, Gliomab-H, GNI-250,EMD-72000, LymphoCide, CMA 676, Monopharm-C, ior egf/r3, ior c5,anti-FLK-2, SMART 1D10, SMART ABL 364, and ImmuRAIT-CEA.

Cancer vaccines are medicaments which are intended to stimulate anendogenous immune response against cancer cells. Currently producedvaccines predominantly activate the humoral immune system (i.e., theantibody dependent immune response). Other vaccines currently indevelopment are focused on activating the cell-mediated immune systemincluding cytotoxic T lymphocytes which are capable of killing tumorcells. Cancer vaccines generally enhance the presentation of cancerantigens to both APCs (e.g., macrophages and dendritic cells) and/or toother immune cells such as T cells, B cells, and NK cells.

Although cancer vaccines can take one of several forms, as discussedinfra, their purpose is to deliver cancer antigens and/or cancerassociated antigens to APCs in order to facilitate the endogenousprocessing of such antigens by APC and the ultimate presentation ofantigen presentation on the cell surface in the context of MHC class Imolecules. One form of cancer vaccine is a whole cell vaccine which is apreparation of cancer cells which have been removed from a subject,treated ex vivo and then reintroduced as whole cells in the subject.Lysates of tumor cells can also be used as cancer vaccines to elicit animmune response. Another form of cancer vaccine is a peptide vaccinewhich uses cancer-specific or cancer-associated small proteins toactivate T cells. Cancer-associated proteins are proteins which are notexclusively expressed by cancer cells (i.e., other normal cells canstill express these antigens). However, the expression ofcancer-associated antigens is generally consistently upregulated withcancers of a particular type. Yet another form of cancer vaccine is adendritic cell vaccine which includes whole dendritic cells which havebeen exposed to a cancer antigen or a cancer-associated antigen invitro. Lysates or membrane fractions of dendritic cells can also be usedas cancer vaccines. Dendritic cell vaccines are able to activate APCsdirectly. Other cancer vaccines include ganglioside vaccines, heat-shockprotein vaccines, viral and bacterial vaccines, and nucleic acidvaccines.

In some embodiments the method also includes administering to thesubject a cytokine A “cytokine” as used herein refers to any of adiverse group of soluble proteins and peptides which act as humoralregulators at nano- to picomolar concentrations and which, either undernormal or pathological conditions, modulate the functional activities ofindividual cells and tissues. These proteins also mediate interactionsbetween cells directly and regulate processes taking place in theextracellular environment. Examples of cytokines include, but are notlimited to interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10,IL-12, IL-15, IL-18; granulocyte-macrophage colony-stimulating factor(GM-CSF); granulocyte colony-stimulating factor (G-CSF); interferonsincluding interferon-alpha (IFN-α), interferon-beta (IFN-β), andinterferon-gamma (IFN-γ); tumor necrosis factor (TNF), transforminggrowth factor-beta (TGF-β; FLT-3 ligand; and CD40 ligand.

Cytokines play a role in directing the T cell response. Helper (CD4+) Tcells orchestrate the immune response of mammals through production ofsoluble factors that act on other immune system cells, including other Tcells. Most mature CD4+ T helper cells express one of two cytokineprofiles: Th1 or Th2. The Th1 subset in mice promotes delayed-typehypersensitivity, cell-mediated immunity, and immunoglobulin classswitching to IgG2a. The Th2 subset in mice induces humoral immunity byactivating B cells, promoting antibody production, and inducing classswitching to IgG1 and IgE. In some embodiments, it is preferred that thecytokine be a Th1 cytokine.

In some embodiments the method according to this aspect of the inventionfurther involves administering to the subject an antibacterialmedicament. An “antibacterial medicament” refers to an agent that killsor inhibits the growth or function of bacteria. Antibacterial agentswhich are effective for killing or inhibiting a wide range of bacteriaare often referred to as broad spectrum antibiotics. Other types ofantibacterial agents are predominantly effective against Gram-positivebacteria or Gram-negative bacteria. These types of antibacterial agentsare frequently referred to as narrow spectrum antibiotics. Otherantibacterial agents which are effective against a single organism ordisease and not against other types of bacteria, are often referred toas limited spectrum antibiotics. Antibacterial agents are sometimesclassified based on their primary mode of action. In general,antibacterial agents are cell wall synthesis inhibitors, cell membraneinhibitors, protein synthesis inhibitors, nucleic acid synthesis orfunctional inhibitors, and competitive inhibitors.

Antibacterial agents useful in the invention include but are not limitedto natural penicillins, semi-synthetic penicillins, clavulanic acid,cephalolsporins, bacitracin, ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin,nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor,cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet,cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam,carbapenems, imipenems, monobactems, eurtreonam, vancomycin, polymyxin,amphotericin B, nystatin, imidazoles, clotrimazole, miconazole,ketoconazole, itraconazole, fluconazole, rifampins, ethambutol,tetracyclines, chloramphenicol, macrolides, aminoglycosides,streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline,minocycline, doxycycline, chlortetracycline, erythromycin,roxithromycin, clarithromycin, oleandomycin, azithromycin,chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin,enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, andtrimethoprim; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine;Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; AmifloxacinMesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefmenoxime Hydrochloride;Cefmetazole; Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; CefotetanDisodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; andZorbamycin.

In some embodiments the method further involves administering to thesubject an antiviral medicament. As used herein, an “antiviralmedicament” refers to a compound which prevents infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g., amantadine), synthesisor translation of viral mRNA (e.g., interferon), replication of viralRNA or DNA (e.g., nucleoside analogues), maturation of new virusproteins (e.g., protease inhibitors), and budding and release of thevirus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine).

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. IFN-α and IFN-βalso induce the expression of Class I and Class II MHC molecules on thesurface of infected cells, resulting in increased antigen presentationfor host immune cell recognition. These interferons are available asrecombinant forms and have been used for the treatment of chronichepatitis B and C infection. At the dosages which are effective forantiviral therapy, interferons sometimes have severe side effects suchas fever, malaise and weight loss.

Immunoglobulin therapy is used for the prevention of viral infection.Immunoglobulin therapy for viral infections is different than bacterialinfections, because rather than being antigen-specific, theimmunoglobulin therapy functions by binding to extracellular virions andpreventing them from attaching to and entering cells which aresusceptible to the viral infection. The therapy is useful for theprevention of viral infection for the period of time that the antibodiesare present in the host. In general there are two types ofimmunoglobulin therapies, normal immune globulin therapy andhyper-immune globulin therapy. Normal immune globulin therapy utilizesan antibody product which is prepared from the serum of normal blooddonors and pooled. This pooled product contains low titers of antibodyto a wide range of human viruses, such as hepatitis A, parvovirus,enterovirus (especially in neonates). Hyper-immune globulin therapyutilizes antibodies which are prepared from the serum of individuals whohave high titers of an antibody to a particular virus. Those antibodiesare then used against a specific virus. Examples of hyper-immuneglobulins include zoster immune globulin (useful for the prevention ofvaricella in immuno-compromised children and neonates), human rabiesimmunoglobulin (useful in the post-exposure prophylaxis of a subjectbitten by a rabid animal), hepatitis B immune globulin (useful in theprevention of hepatitis B virus, especially in a subject exposed to thevirus), and RSV immune globulin (useful in the treatment of respiratorysyncitial virus infections).

Thus, antiviral medicaments useful in the invention include but are notlimited to immunoglobulins, amantadine, interferon, nucleosideanalogues, and protease inhibitors. Specific examples of antiviralagents include but are not limited to Acemannan; Acyclovir; AcyclovirSodium; Adefovir; Alovudine; Alvircept Sudotox; AmantadineHydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine;Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime;Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine;Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; GanciclovirSodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; MemotineHydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; SomantadineHydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride;Trifluridine; Valacyclovir Hydrochloride; Vidarabine; VidarabinePhosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine;Zidovudine; and Zinviroxime.

In some embodiments the method according to this aspect of the inventionfurther involves administering to the subject an antifungal medicament.An “antifungal medicament” is an agent that kills or inhibits the growthor function of infective fungi. Anti-fungal medicaments are sometimesclassified by their mechanism of action. Some anti-fungal agentsfunction as cell wall inhibitors by inhibiting glucose synthase. Theseinclude, but are not limited to, basiungin/ECB. Other antifungal agentsfunction by destabilizing membrane integrity. These include, but are notlimited to, imidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other antifungal agents function bybreaking down chitin (e.g., chitinase) or immunosuppression (501 cream).

Thus, the antifungal medicaments useful in the invention include but arenot limited to imidazoles, 501 cream, and Acrisorcin, Ambruticin,Amorolfine, Amphotericin B, Azaconazole, Azaserine, Basifungin, BAY38-9502, Bifonazole, Biphenamine Hydrochloride, Bispyrithione Magsulfex,Butenafine, Butoconazole Nitrate, Calcium Undecylenate, Candicidin,Carbol-Fuchsin, Chitinase, Chlordantoin, Ciclopirox, Ciclopirox Olamine,Cilofungin, Cisconazole, Clotrimazole, Cuprimyxin, Denofungin,Dipyrithione, Doconazole, Econazole, Econazole Nitrate, Enilconazole,Ethonam Nitrate, Fenticonazole Nitrate, Filipin, FK 463, Fluconazole,Flucytosine, Fungimycin, Griseofulvin, Hamycin, Isoconazole,Itraconazole, Kalafungin, Ketoconazole, Lomofungin, Lydimycin,Mepartricin, Miconazole, Miconazole Nitrate, MK 991, Monensin, MonensinSodium, Naftifine Hydrochloride, Neomycin Undecylenate, Nifuratel,Nifurmerone, Nitralamine Hydrochloride, Nystatin, Octanoic Acid,Orconazole Nitrate, Oxiconazole Nitrate, Oxifungin Hydrochloride,Parconazole Hydrochloride, Partricin, Potassium Iodide, Pradimicin,Proclonol, Pyrithione Zinc, Pyrrolnitrin, Rutamycin, SanguinariumChloride, Saperconazole, Scopafungin, Selenium Sulfide, Sertaconazole,Sinefungin, Sulconazole Nitrate, Terbinafine, Terconazole, Thiram,Ticlatone, Tioconazole, Tolciclate, Tolindate, Tolnaftate, Triacetin,Triafungin, UK 292, Undecylenic Acid, Viridofulvin, Voriconazole, ZincUndecylenate, and Zinoconazole Hydrochloride.

In some embodiments the method further involves administering to thesubject an antiparasitic medicament. An “antiparasitic medicament”refers to an agent that kills or inhibits the growth or function ofinfective parasites. Examples of antiparasitic medicaments, alsoreferred to as parasiticides, useful in the invention include but arenot limited to albendazole, amphotericin B, benznidazole, bithionol,chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine,diethylcarbamazine, diloxanide furoate, doxycycline, eflornithine,furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin,mebendazole, mefloquine, meglumine antimoniate, melarsoprol,metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine,paromomycin, pentamidine isethionate, piperazine, praziquantel,primaquine phosphate, proguanil, pyrantel pamoate,pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl,quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium(sodium antimony gluconate), suramin, tetracycline, thiabendazole,tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide, some ofwhich are used alone or in combination with others.

Certain aspects of the invention involve methods which inhibit T-celltrafficking into selected tissues.

Accordingly, in another aspect the invention provides a method forreducing T-cell trafficking into a target tissue of a subject. Themethod according to this aspect involves administering to the subject aTIM-3 ligand-binding molecule in an effective amount to reduce T-celltrafficking to a target tissue of the subject. A “TIM-3 ligand-bindingmolecule” as used herein refers to any molecule, including TIM-3, thatbinds to TIM-3 ligand.

In addition to TIM-3 itself, the TIM-3 ligand-binding molecule can be asmall molecule, a polypeptide, an antibody or a fragment of an antibody,a polynucleotide, a carbohydrate including a polysaccharide, a lipid, adrug, as well as mimics, derivatives, and combinations thereof. TheTIM-3 ligand-binding molecule can be found in nature or it can bederived or synthesized using suitable in vitro and synthetic methodsknown by those of skill in the art. For example, the TIM-3ligand-binding molecule can be a small molecule that is identifiedthrough screening a library of small molecules for the ability to bindto TIM-3 ligand. As another example, the TIM-3 ligand-binding moleculecan be generated and identified using phage display of peptides.

In some embodiments the TIM-3 ligand-binding molecule is a solubleTIM-3. “Soluble TIM-3” refers to any form of TIM-3, including functionalvariants of TIM-3, that is dissociated from cell membrane. Soluble TIM-3can be a C-terminal truncated form of full-length TIM-3 or atransmembrane-deleted version of TIM-3. In some embodiments the TIM-3ligand-binding molecule includes a single domain of the extracellularregion of TIM-3, i.e., the IgV domain or the mucin domain. In oneembodiment the soluble TIM-3 is an alternatively spliced variant offull-length TIM-3, which includes the IgV domain and intracellularregion but not the mucin domain or transmembrane region. In someembodiments the TIM-3 ligand-binding molecule includes an extracellularregion of TIM-3.

In some embodiments the soluble TIM-3 is a fusion protein including atleast one domain of an extracellular region of TIM-3 and a constantheavy chain or portion thereof of an immunoglobulin. In one embodimentsoluble TIM-3 refers to a fusion protein that includes at least onedomain of an extracellular domain of TIM-3 and another polypeptide. Inone embodiment the soluble TIM-3 is a fusion protein including theextracellular region of TIM-3 covalently linked, e.g., via a peptidebond, to an Fc fragment of an immunoglobulin such as IgG; such a fusionprotein typically is a homodimer. In one embodiment the soluble TIM-3 isa fusion protein including just the IgV domain of the extracellularregion of TIM-3 covalently linked, e.g., via a peptide bond, to an Fcfragment of an immunoglobulin such as IgG; such a fusion proteintypically is a homodimer. As is well known in the art, an Fc fragment isa homodimer of two partial constant heavy chains. Each constant heavychain includes at least a CH1 domain, the hinge, and CH2 and CH3domains. Each monomer of such an Fc fusion protein includes anextracellular region of TIM-3 linked to a constant heavy chain orportion thereof (e.g., hinge, CH2, CH3 domains) of an immunoglobulin.The constant heavy chain in some embodiments will include part or all ofthe CH1 domain that is N-terminal to the hinge region of immunoglobulin.In other embodiments the constant heavy chain will include the hinge butnot the CH1 domain. In yet other embodiments the constant heavy chainwill exclude the hinge and the CH1 domain, e.g., it will include onlythe CH2 and CH3 domains of IgG.

In some embodiments the subject is in need of treatment for anautoimmune disease of the target tissue. “Autoimmune disease” is a classof diseases in which a subject's own antibodies react with host tissueor in which immune effector T cells are autoreactive to endogenousself-peptides and cause destruction of tissue. Thus an immune responseis mounted against a subject's own antigens, referred to asself-antigens. Autoimmune diseases include but are not limited torheumatoid arthritis, Crohn's disease, multiple sclerosis, systemiclupus erythematosus (SLE), autoimmune encephalomyelitis, myastheniagravis (MG), Hashimoto's thyroiditis, Goodpastures syndrome, pemphigus(e.g., pemphigus vulgaris), Grave's disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polymyositis,pernicious anemia, idiopathic Addison's disease, autoimmune-associatedinfertility, glomerulonephritis (e.g., crescentic glomerulonephritis,proliferative glomerulonephritis), bullous pemphigoid, Sjögren'ssyndrome, insulin resistance, and autoimmune diabetes mellitus (type 1diabetes mellitus; insulin-dependent diabetes mellitus). Recentlyautoimmune disease has been recognized also to encompass atherosclerosisand Alzheimer's disease.

A “self-antigen” as used herein refers to an antigen of a normal hosttissue. Normal host tissue does not include cancer cells. Thus an immuneresponse mounted against a self-antigen, in the context of an autoimmunedisease, is an undesirable immune response and contributes todestruction and damage of normal tissue, whereas an immune responsemounted against a cancer antigen is a desirable immune response andcontributes to destruction of the tumor or cancer.

In yet another aspect the invention provides a method for treating orpreventing asthma or allergy. The method according to this aspect of theinvention involves increasing activity or expression of TIM-3 in a Tcell of a subject to treat or prevent asthma or allergy.

As used herein, “asthma” shall refer to a disorder of the respiratorysystem characterized by inflammation and narrowing of the airways andincreased reactivity of the airways to inhaled agents. Asthma isfrequently, although not exclusively, associated with atopic or allergicsymptoms.

As used herein, “allergy” shall refer to acquired hypersensitivity to asubstance (allergen). Allergic conditions include eczema, allergicrhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) andfood allergies, and other atopic conditions. A “subject having anallergy” is a subject that has or is at risk of developing an allergicreaction in response to an allergen. An “allergen” refers to a substancethat can induce an allergic or asthmatic response in a susceptiblesubject. The list of allergens is enormous and can include pollens,insect venoms, animal dander, dust, fungal spores and drugs (e.g.,penicillin).

Examples of natural animal and plant allergens include proteins specificto the following genuses: Canine (Canis familiaris); Dermatophagoides(e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia(Ambrosia artemiisfolia; Lolium (e.g., Lolium perenne or Loliummultiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternariaalternate); Alder; Alnus (Alnus gultinosa); Betula (Betula verrucosa);Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisiavulgaris); Plantago (e.g., Plantago lanceolata); Parietaria (e.g.,Parietaria officinalis or Parietaria judaica); Blattella (e.g.,Blattella germanica); Apis (e.g., Apis multiflorum); Cupressus (e.g.,Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa);Juniperus (e.g., Juniperus sabinoides, Juniperus virginiana, Juniperuscommunis and Juniperus ashei); Thuya (e.g., Thuya orientalis);Chamaecyparis (e.g., Chamaecyparis obtusa); Periplaneta (e.g.,Periplaneta americana); Agropyron (e.g., Agropyron repens); Secale(e.g., Secale cereale); Triticum (e.g., Triticum aestivum); Dactylis(e.g., Dactylis glomerate); Festuca (e.g., Festuca elation); Poa (e.g.,Poa pratensis or Poa compressa); Avena (e.g., Avena sativa); Holcus(e.g., Holcus lanatus); Anthoxanthum (e.g., Anthoxanthum odoratum);Arrhenatherum (e.g., Arrhenatherum elatius); Agrostis (e.g., Agrostisalba); Phleum (e.g., Phleum pratense); Phalaris (e.g., Phalarisarundinacea); Paspalum (e.g., Paspalum notatum); Sorghum (e.g., Sorghumhalepensis); and Bromus (e.g., Bromus inermis).

According to a further aspect, the invention provides a method fortreating a Th2-mediated disorder in a subject. The method involvesexpressing TIM-3 on the surface of Th2 cells of a subject having aTh2-mediated disorder in an amount effective to treat the Th2-mediateddisorder. A “Th2-mediated disorder” as used herein refers to a diseasethat is associated with the development of a Th2 immune response. A “Th2immune response” as used herein refers to the induction of at least oneTh2-cytokine or a Th2-antibody. In preferred embodiments more than oneTh2-cytokine or Th2-antibody is induced. Thus a Th2-mediated disease isa disease associated with the induction of a Th2 response and refers tothe partial or complete induction of at least one Th2-cytokine orTh2-antibody or an increase in the levels of at least one Th2-cytokineor Th2-antibody. These disorders are known in the art and include forinstance, but are not limited to, atopic conditions, such as asthma andallergy, including allergic rhinitis, gastrointestinal allergies,including food allergies, eosinophilia, conjunctivitis,glomerulonephritis, certain pathogen susceptibilities such as helminthic(e.g., leishmaniasis) and certain viral infections, including humanimmunodieficiency virus (HIV), and certain bacterial infections,including tuberculosis and lepromatous leprosy.

In contrast to a Th2-mediated disorder, a “Th1-mediated disorder” asused herein refers to a disease that is associated with the developmentof a Th1 immune response. A “Th1 immune response” as used herein refersto the induction of at least one Th1-cytokine or a Th1-antibody. Inpreferred embodiments more than one Th1-cytokine or Th1-antibody isinduced. Thus a Th1-mediated disease is a disease associated with theinduction of a Th1 response and refers to the partial or completeinduction of at least one Th1-cytokine or Th1-antibody or an increase inthe levels of at least one Th1-cytokine or Th1-antibody. These disordersare known in the art and include for instance, but are not limited to,autoimmune (especially organ-specific) disease, psoriasis, Th1inflammatory disorders, infection with extracellular parasites (e.g.,response to helminths), solid organ allograft rejection (e.g., acutekidney allograft rejection), symptoms associated with hepatitis B (HBV)infection (e.g., HBV acute phase or recovery phase), chronic hepatitis C(HCV) infection, insulin-dependent diabetes mellitus (IDDM), multiplesclerosis (MS), subacute lymphocytic thyroiditis (“silent thyroiditis”),Crohn's disease, primary biliary cirrhosis, primary sclerosingcholangitis, sarcoidosis, atherosclerosis, acute graft-versus-hostdisease (GvHD), glomerulonephritis, anti-glomerular basement membranedisease, Wegener's granulomatosis, inflammatory myopathies, Sjögren'ssyndrome, Behçet's syndrome, rheumatoid arthritis, Lyme arthritis, andunexplained recurrent abortion. In some embodiments the Th1-mediateddisorder is selected from the group consisting of atherosclerosis,infection with extracellular parasites, symptoms associated withhepatitis B (HBV) infection (e.g., HBV acute phase or recovery phase),chronic hepatitis C (HCV) infection, silent thyroiditis, primary biliarycirrhosis, primary sclerosing cholangitis, glomerulonephritis,anti-glomerular basement membrane disease, Wegener's granulomatosis,inflammatory myopathies, Sjögren's syndrome, Behçet's syndrome,rheumatoid arthritis, and unexplained recurrent abortion.

According to another aspect, the invention provides a method forpromoting APC activation. The method according to this aspect involvescontacting a T cell with a TIM-3-binding molecule, and contacting an APCwith the T cell to activate the APC.

An “antigen-presenting cell” or, equivalently, an “APC” as used hereinrefers to a specialized cell, either belonging to the immune system orcapable of interacting with a cell belonging to the immune system, thatpresents on its cell surface a complex between an antigen and a majorhistocompatability complex (MHC) molecule. In addition to presentingantigen in the context of self (i.e., self-MHC) to antigen-specificlymphocytes, APCs frequently also provide additional costimulatorysignals to lymphocytes so engaged, through cognate interactions betweenadditional cell surface receptor/counter-receptor pairs and throughsecreted products, e.g., cytokines and chemokines APCs are believed toinclude mononuclear phagocytes (e.g., monocyte/macrophages), Blymphocytes, dendritic cells, Langerhans cells, and certain endothelialcells. In certain embodiments an APC is a macrophage. In certainembodiments an APC is a dendritic cell. It has been discovered accordingto the present invention, for example, that certain APCs, includingmacrophages and dendritic cells, express TIM-3 ligand on their cellsurface.

According to yet another aspect of the invention, a method is providedfor inhibiting APC activation. The method involves contacting an APCwith an an effective amount of an agent that reduces activity orexpression of TIM-3 to inhibit activation of the APC. An “agent thatreduces activity or expression of TIM-3” as used herein refers to anagent that reduces the function of TIM-3 either by effectively blockinginteraction of TIM-3 with its ligand or by interfering with expressionof TIM-3. Such agents can take the form of TIM-3-binding molecules,TIM-3 ligand-binding molecules, and antisense to TIM-3.

According to another aspect the invention further provides a method fortreating or preventing intracellular infections. The method according tothis aspect involves promoting macrophage activation by contacting aTIM-3 ligand on the macrophage with a TIM-3 expressing cell. An“intracellular infection” as used herein refers to an infection by aninfectious organism or agent wherein the infectious organism or agentsurvives and replicates within a cell of the infected host. A number ofbacteria, fungi, parasites, and all viruses are involved inintracellular infections. Intracellular bacteria such as Listeriamonocytogenes and various mycobacteria, including M. tuberculosis, areresistant to degradation within macrophages and are therefore capable ofsurviving and replicating within phagocytes. Such intracellular bacterianormally pose a special challenge to the immune system's ability toeradicate the infecting organisms. Another important example of anintracellular infection is that involving the obligate intracellularprotozoa of the genus Leishmania.

When administered to a subject, the TIM-3-binding molecule and TIM-3ligand-binding molecule of the present invention are administered inpharmaceutically acceptable preparations. Such preparations mayroutinely contain pharmaceutically acceptable concentrations of salt,buffering agents, preservatives, compatible carriers, supplementaryagents such as adjuvants and cytokines, and optionally other therapeuticagents. Thus, “cocktails” including the TIM-3-binding molecule and TIM-3ligand-binding molecule and optional supplementary agents arecontemplated. The supplementary agents themselves can be conjugated toTIM-3-binding molecule or TIM-3 ligand-binding molecule to enhancedelivery of the supplementary agents.

The preferred amount of TIM-3-binding molecule or TIM-3 ligand-bindingmolecule in all pharmaceutical preparations made in accordance with thepresent invention should be a therapeutically effective amount thereofwhich is also a medically acceptable amount thereof. Actual dosagelevels of TIM-3-binding molecule or TIM-3 ligand-binding molecule in thepharmaceutical compositions of the present invention can be varied so asto obtain an amount of TIM-3-binding molecule or TIM-3 ligand-bindingmolecule which is effective to achieve the desired therapeutic responsefor a particular patient, pharmaceutical composition of TIM-3-bindingmolecule or TIM-3 ligand-binding molecule, and mode of administration,without being toxic to the patient.

The selected dosage level and frequency of administration will dependupon a variety of factors including the route of administration, thetime of administration, the rate of excretion of the therapeuticagent(s) including TIM-3-binding molecule and TIM-3 ligand-bindingmolecule, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with TIM-3-binding molecule or TIM-3ligand-binding molecule, the age, sex, weight, condition, general healthand prior medical history of the patient being treated and the likefactors well known in the medical arts. For example, the dosage regimenis likely to vary with pregnant women, nursing mothers and childrenrelative to healthy adults.

A physician having ordinary skill in the art can readily determine andprescribe the therapeutically effective amount of the pharmaceuticalcomposition required. For example, the physician could start doses ofTIM-3-binding molecule or TIM-3 ligand-binding molecule employed in thepharmaceutical composition of the present invention at levels lower thanthat required to achieve the desired therapeutic effect and graduallyincrease the dosage until the desired effect is achieved.

For use in therapy, the TIM-3-binding molecule and TIM-3 ligand-bindingmolecule can be formulated as pharmaceutical compositions. Thepharmaceutical compositions of the invention contain an effective amountof TIM-3-binding molecule or TIM-3 ligand-binding molecule, andoptionally another medicament, in a pharmaceutically acceptable carrier.The term “pharmaceutically acceptable carrier” means one or morecompatible solid or liquid fillers, dilutants or encapsulatingsubstances which are suitable for administration to a human or othervertebrate animal. The term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The components of thepharmaceutical compositions also are capable of being commingled withthe TIM-3-binding molecule or TIM-3 ligand-binding molecule of thepresent invention, and with each other, in a manner such that there isno interaction which would substantially impair the desiredpharmaceutical efficacy.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer R (1990) Science249:1527-33, which is incorporated herein by reference.

For use in therapy, an effective amount of the TIM-3-binding molecule orTIM-3 ligand-binding molecule can be administered to a subject by anymode that delivers the TIM-3-binding molecule or TIM-3 ligand-bindingmolecule either systemically or to the desired surface, e.g., skin ormucosa. “Administering” the pharmaceutical composition of the presentinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited to oral,parenteral, intravenous, intramuscular, intracutaneous, subcutaneous,intradermal, subdermal, transdermal, topical, intranasal, intratracheal,inhalation, ocular, vaginal, and rectal. In certain preferredembodiments the preferred route of administration is systemic orintravenous.

The TIM-3-binding molecule or TIM-3 ligand-binding molecule, when it isdesirable to deliver them systemically, may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The TIM-3-binding molecule or TIM-3 ligand-bindingmolecule may take such forms as suspensions, solutions or emulsions inoily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the TIM-3-binding molecule or TIM-3 ligand-bindingmolecule in water-soluble form. Additionally, suspensions of theTIM-3-binding molecule or TIM-3 ligand-binding molecule may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the TIM-3-binding molecule or TIM-3 ligand-bindingmolecule may be in powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

For oral administration, the TIM-3-binding molecule or TIM-3ligand-binding molecule can be formulated readily by combining theactive compound(s) with pharmaceutically acceptable carriers well knownin the art. Such carriers enable the compounds of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject tobe treated. Pharmaceutical preparations for oral use can be obtained assolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the crosslinkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. Optionally the oral formulations may also be formulatedin saline or buffers for neutralizing internal acid conditions or may beadministered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the TIM-3-binding molecule or TIM-3ligand-binding molecule for use according to the present invention maybe conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch. Techniques for preparing aerosol delivery systemsare well known to those of skill in the art. Generally, such systemsshould utilize components which will not significantly impair thebiological properties of the therapeutic, such as the immunomodulatorycapacity of the TIM-3-binding molecule or TIM-3 ligand-binding molecule(see, for example, Sciarra and Cutie, “Aerosols,” in Remington'sPharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporatedby reference). Those of skill in the art can readily determine thevarious parameters and conditions for producing aerosols without resortto undue experimentation.

In some embodiments, topical administration is preferred. For topicaladministration to the skin, the TIM-3-binding molecule or TIM-3ligand-binding molecule according to the invention may be formulated asointments, gels, creams, lotions, or as a transdermal patch foriontophoresis. One method for accomplishing topical administrationincludes transdermal administration, such as by iontophoresis.Iontophoretic transmission can be accomplished by using commerciallyavailable patches which deliver a compound continuously through unbrokenskin for periods of hours to days to weeks, depending on the particularpatch. This method allows for the controlled delivery of theTIM-3-binding molecule or TIM-3 ligand-binding molecule and/oradditional medicament through the skin in relatively highconcentrations. One example of an iontophoretic patch is the LECTROPATCH™ sold by General Medical Company of Los Angeles, Calif. The patchprovides dosages of different concentrations which can be continuouslyor periodically administered across the skin using electronicstimulation of reservoirs containing the TIM-3-binding molecule or TIM-3ligand-binding molecule and/or additional medicament.

For topical administration to the skin, ointments, gels creams, andlotions can be formulated with an aqueous or oily base alone or togetherwith suitable thickening and/or gelling agents. Lotions can beformulated with an aqueous or oily base and, typically, further includeone or more emulsifying agents, stabilizing agents, dispersing agents,suspending agents, thickening agents, or coloring agents. (See, e.g.,U.S. Pat. No. 5,563,153, entitled “Sterile Topical Anesthetic Gel”,issued to Mueller, D., et al., for a description of a pharmaceuticallyacceptable gel-based topical carrier.) The ointments, gels, creams, orlotions can optionally be formulated to include sunscreen compounds,fragrance, moisturizing agents, or coloring agents. Sunscreen compoundsinclude those organic and inorganic materials employed to blockultraviolet light. Illustrative organic sunscreen compounds arederivatives of p-aminobenzoic acid (PABA), cinnamate, salicylate,benzophenones, anthranilates, dibenzoylmethanes, and camphores; examplesor inorganic sunscreen compounds include zinc oxide and titaniumdioxide. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone) can be used. Octylmethoxycinnamate and 2-hydroxy-4-methoxy benzophenone are commerciallyavailable under the trademarks Parsol MCX and Benzophenone-3,respectively. Other examples include 2-phenylbenzimidazole-5-sulfonicacid (commercially available as Eusolex 232 from Rona), andoctyldimethyl p-amino benzoic acid (octyl dimethyl PABA commerciallyavailable from Haarmann & Reimer). The exact amount of sunscreenemployed in the emulsions can vary depending upon the degree ofprotection desired from the sun's UV radiation.

The TIM-3-binding molecule or TIM-3 ligand-binding molecule may also beformulated in rectal or vaginal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

In addition to the formulations described previously, the TIM-3-bindingmolecule or TIM-3 ligand-binding molecule may also be formulated as adepot preparation. Such long-acting formulations may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also can include suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

The TIM-3-binding molecule or TIM-3 ligand-binding molecule andadditional medicament may be administered per se (neat) or in the formof a pharmaceutically acceptable salt. When used in medicine the saltsshould be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof. Such salts include, but are not limited to,those prepared from the following acids: acetic, benzene sulphonic,citric, formic, hydrobromic, hydrochloric, maleic, malonic, methanesulphonic, naphthalene-2-sulphonic, nitric, p-toluene sulphonic,phosphoric, salicylic, succinic, sulfuric, and tartaric. Also, suchsalts can be prepared as alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical compositions may be conveniently presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. All methods include the step of bringing the TIM-3-bindingmolecule or TIM-3 ligand-binding molecule into association with acarrier which constitutes one or more accessory ingredients. In general,the compositions are prepared by uniformly and intimately bringing theTIM-3-binding molecule or TIM-3 ligand-binding molecule into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the TIM-3-binding molecule or TIM-3 ligand-bindingmolecule of the invention, increasing convenience to the subject and thephysician. Many types of release delivery systems are available andknown to those of ordinary skill in the art. They include polymer basedsystems such as polytactic and polyglycolic acid, polyanhidrides andpolycaprolactone; wax coatings, compressed tablets using conventionalbinders and excipients, and the like. Bioadhesive polymer systems toenhance delivery of a material to the intestinal epithelium are knownand described in published PCT application WO 93/21906. Capsules fordelivering agents to the intestinal epithelium also are described inpublished PCT application WO 93/19660.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example include metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES Example 1 Generation of Th1-Specific mAb

To Identify New Th1-Specific Cell surface proteins, Lewis and Lou/M ratswere immunized with Th1 T cell clones and lines, including theestablished Th1-specific clone AE7 and in vitro differentiated Th1 celllines derived from 5B6 (Waldner H et al. (2000) Proc Natl Acad Sci USA97:3412-17) and DO11.10 TCR transgenic mice. Lewis and Lou/M female rats(Harlan Sprague-Dawley, Inc.) were immunized three times by acombination of subcutaneous (s.c.) injection with either 1-5×10⁷Th1-polarized T cell clones and/or lines. The rats were boosted and fourdays later spleen cells were fused with myeloma cells (ATCC No. CRL8006)using polyethylene glycol 1450 and selection in HAT medium. Kohler G etal. (1975) Nature 256:495-97. A panel of approximately 20,000 monoclonalantibodies (mAb) was generated. The supernatants from the fusion platewells were screened by flow cytometry on Th1 and Th2 T cells. Allhybridoma wells that gave a positive shift on Th1, but not Th2, T cellclones were expanded and subcloned twice by limiting dilution. Two ofthe mAbs that selectively stained Th1 cells, 8B.2C12 and 25F.1D6, werefurther characterized. These mAbs recognize a cell surface proteinpresent on established CD4+ Th1 cells and CD8+ Tc1 cells, but absent onCD4+ Th2 cells and CD8+ Tc2 cells (FIG. 1A).

Example 2 Cloning of TIM-3

A eukaryotic expression library was constructed using mRNA from the AE7Th1 clone and the pAXF vector. Library screening was carried out byexpression cloning according to the method developed by Seed. Seed B etal. (1987) Proc Natl Acad Sci USA 84:3365-69. Immunoselected individualplasmids were transfected into COS cells followed by indirectimmunofluorescence staining with anti-TIM-3 antibody. Positive cloneswere sequenced. By this method, a murine cDNA was identified thatencodes a type I membrane protein of 281 amino acids with the followingregions: an extracellular domain consisting of an IgV-like domainfollowed by a mucin-like domain consisting of 31% serine and threonineresidues; a transmembrane region; and a cytoplasmic region (FIG. 1B).This gene product expressed by the cDNA was named TIM-3, T cellImmunoglobulin and Mucin domain containing molecule. The cytoplasmicregion contains six tyrosines, one of which is part of a tyrosinephosphorylation motif, RSEENIY (SEQ ID NO:7). The extracellular domaincontains four sites for N-linked glycosylation and five sites forO-linked glycosylation.

The human homologue of TIM-3 was identified through genomic databasesearches and reverse transcriptase-polymerase chain reaction (RT-PCR).It has 63% amino acid identity to murine TIM-3 overall with 77% identityin the cytoplasmic domain, including conservation of the tyrosinephosphorylation site (FIG. 1B). Database searches revealed that TIM-3 isrelated to Kidney Injury Molecule-1 (KIM-1)/HAVcr-1, the receptor forhuman hepatitis A virus, and TIM-2. All three family members (KIM-1,TIM-2 and TIM-3) have a similar Ig/mucin structure, and their genes arelocated on human chromosome 5q33.2 and mouse chromosome 11. McIntire J Jet al. (2001) Nat Immunol 2:1109-1116.

Example 3 Expression of TIM-3 In Vitro

A TIM-3 cDNA expression construct was used to stably transfect ChineseHamster Ovary (CHO) cells. Flow cytometry and immunoprecipitationanalysis of the stable transfectants confirmed that the cloned proteinis TIM-3 and that it is expressed at the cell surface (FIG. 1C).

Expression of this gene in various cell lines (Th1, Th2, dendritic cell,macrophage and B cells) and SJL T, B and CD11b+ cells was determined byquantitative RT-PCR. TIM-3 transcripts were present at the highest levelonly in Th1 cells, although in vivo-derived CD11b+ cells also showedlow-level expression (FIG. 1D). Flow cytometric analysis usinganti-TIM-3 antibodies confirmed that TIM-3 is not expressed on naïve Tcells, B cells, macrophages or dendritic cells. These data suggest thatthe molecule recognized by these antibodies is expressed mainly ondifferentiated Th1 or Tc1 cells and not on other hematopoietic celltypes, at least at the protein level.

Example 4 Kinetics of TIM-3 Expression

To examine the kinetics of TIM-3 protein expression during T celldifferentiation, naïve DO11.10 TCR transgenic T cells were activated invitro under Th1- or Th2-polarizing conditions. After each round ofrestimulation, Th1 and Th2 cells were stimulated with PMA/ionomycin forthe induction of cytokines and then stained with mAbs to TIM-3 and CD4,and cytokine expression was detected by intracellular staining. In thisexperiment TIM-3 was not observed on Th2 cells but was detectable on Th1cells after the third round of in vitro polarization (FIG. 2). TIM-3 istherefore expressed at a late stage of T-cell differentiation,suggesting that TIM-3 may not contribute to T-cell differentiation perse but can play a role in the trafficking and/or effector functions ofTh1 cells.

Example 5 Expression of TIM-3 in EAE

Expression of TIM-3 was then examined during the development of EAE, aTh1-mediated autoimmune disease of the CNS. EAE-susceptible SJL micewere immunized with the encephalitogenic proteolipid protein (PLP)139-151 peptide HSLGKWLGHPDKF (SEQ ID NO:8; Quality ControlledBiochemicals). Female SJL mice (4-8 weeks old) (The Jackson Laboratory)were injected s.c. in each flank with 50 μg of PLP 139-151 peptideemulsified in complete Freund's adjuvant (CFA; Difco) supplemented with400 μg of Mycobacterium tuberculosis (Difco) for the induction of EAE.Mice were examined daily for signs of EAE, which were graded as follows:flaccid tail, 1; uneven gait and impaired righting reflex, 2; totalhindlimb paralysis, 3; fore- and hindlimb paralysis, 4; and moribund, 5.Mice were scored for disease and sacrificed at various time pointsfollowing immunization, and spleen, brain and lymph nodes were removedand tested for the expression of TIM-3.

Real-time reverse transcriptase-quantitative polymerase chain reaction(RT-QPCR) was performed using the Taqman® strategy (Applied Biosystems).Total RNA was isolated by TRIzol and treated with DNase I. RNA wasconverted to cDNA by reverse transcription, and 10 ng of cDNA was usedfor Taqman® PCR. The expression levels of TIM-3 and internal referenceglyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured bymultiplex PCR using probes labeled with 6-carboxyfluorescein (FAM) orVIC® (Applied Biosystems), respectively. TIM-3 primers and probes weredesigned in the 3′ UTR of the 2.8 kb transcript using Primer Expressv1.0 software (Applied Biosystems). The GAPDH primer/probe set waspurchased from Applied Biosystems. The following TIM-3-specific primersand probe were employed for TIM-3:

(SEQ ID NO: 9) forward primer: CAAGCCGGTGGACCTCAGT; (SEQ ID NO: 10)reverse primer: AGATGGGAGCCAGCACAG; (SEQ ID NO: 11) probe:AGCTGCCTGCCCAGTGCCCTT.

The simultaneous measurement of TIM-3—FAM and GAPDH—VIC permittednormalization of the amount of cDNA added per sample. PCRs wereperformed using the Taqman® Universal PCR Master Mix and the ABI PRISM7700 Sequence Detection System. A comparative threshold cycle (C_(T))was used to determine gene expression relative to the no-tissue control(calibrator). Hence steady-state mRNA levels were expressed as an n-folddifference relative to the calibrator. For each sample, the TIM-3 C_(T)value was normalized using the formula ΔC=C_(TTIM-3)-C_(TGAPDH). Todetermine relative expression levels, the following formula was used:ΔΔC_(T)=Δ_(CT(1)sample)-Δ_(CT(1)calibrator), and the value used to graphrelative TIM-3 expression was calculated using the expression 2^(−ΔΔCT).

FIG. 3A shows expression of TIM-3 RNA relative to control GAPDHexpression. Using quantitative RT-PCR, TIM-3 mRNA was demonstrated to beupregulated in the lymph node following immunization, with expressionpeaking at day 7 post-immunization, just prior to EAE onset. Expressionin the lymph nodes was then downregulated as the disease progressed. Inthe brain, TIM-3 mRNA expression steadily increased and peaked at thebeginning of the disease (around day 10-13). TIM-3 mRNA expression wasthen downregulated to near basal levels at the maximal disease score(FIG. 3A). This expression pattern was confirmed at the protein level byflow cytometric (FACS) analysis (FIG. 3B). On day 10, cells from brain,spleen and lymph nodes were stained with mAbs to TIM-3, CD4, CD8, CD11b,CD19 and B220. Histograms represent TIM-3 expression on different cellpopulations (dotted line, isotype control; solid line, specificstaining) Whereas TIM-3 was expressed on very few CD4+ T cells (<2%) inthe periphery following immunization, TIM-3 was specifically expressedon the majority of CD4+ and CD8+ T cells present in the CNS at the onsetof clinical signs of disease (FIG. 3B). The number of TIM-3+ T cellsdecreased in the CNS as the disease progressed. These data suggest thatTIM-3 is expressed in vivo on T cells and plays an important role in theinitiation of EAE. These data further suggest that TIM-3 expression ondifferentiated Th1 cells allows Th1 cells preferentially to infiltrateCNS tissue and that TIM-3-expressing cells have an advantage forexpansion in the CNS.

Example 6 Hyperacute EAE Following Administration of Anti-TIM-3

To further study the function of TIM-3 in vivo, the effect of anti-TIM-3antibody administration on the development of EAE was tested. SJL micepreviously immunized with PLP 139-151 peptide were administeredanti-TIM-3 antibody or isotype-matched control rat Ig (rIgG2a) andobserved for the development of EAE. Female SJL mice (4-8 weeks old)(The Jackson Laboratory) were injected s.c. in each flank with 25-75 μgof PLP 139-151 peptide in CFA supplemented with 400 μg of M.tuberculosis. Each mouse was also injected i.v. with 100 ng of pertussistoxin (List Biological Laboratories) in 0.1 mL of PBS. Mice wereinjected intraperitoneally (i.p.) every other day beginning on day 0 andcontinuing for 10-16 days with either 100 μg anti-TIM-3 (endotoxinactivity of 0.2 to 0.8 EU/mg) or 100 μg control rIgG or PBS. Mice wereexamined daily for signs of EAE, which were graded as described above.At the peak of the disease or at the end of the experiment, brains andspinal cords were removed and fixed in 10% formalin and examinedhistopathologically for inflammation and demyelination.

Mice treated with anti-TIM-3 developed a hyperacute and atypical EAE,with increased weight loss, malaise, ataxia and paralysis of theforelimbs, without hindlimb paralysis and while sometimes retaining tailtone. While disease onset was normal in the group treated withanti-TIM-3 antibody, disease progression was accelerated and resulted insignificantly more severe clinical disease and increased mortalitycompared to the control group treated with rIgG2a (Table 1).

Histologically, the anti-TIM-3-treated mice generally showed typicalfindings of acute EAE, but with increased numbers of inflammatory fociboth in the meninges and parenchyma compared to the controlrIgG2a-treated group (Table 1). Anti-TIM-3-treated animals that weresacrificed immediately after the onset of clinical signs showed apreponderance of neutrophils and mononuclear cells in the CNSinflammatory infiltrates with focal perivascular fibrin deposition,indicating vascular damage and a hyperacute inflammatory response (FIG.4A, 4B). Furthermore, anti-TIM-3-treated animals that were sacrificed at30 days showed more extensive demyelinating lesions compared torIgG2a-treated animals (FIG. 4C, 4D versus FIG. 4E, 4F). Thedemyelinating lesions in anti-TIM-3-treated mice were filled withactivated macrophages with detectable phagocytosed myelin fragments(FIG. 4D, inset). It has previously been shown that activatedmacrophages are the primary cells responsible for demyelination in EAE(Gordon E J et al. (1995) J Neuroimmunol 62:153-60), and that depletionof activated macrophages leads to an inhibition of EAE. Tran E H et al.(1998) J Immunol 161:3767-75. Thus, it is the applicants' belief thatactivated macrophages induced by anti-TIM-3 antibody treatment areresponsible for the hyperacute disease phenotype and enhancedinflammation and demyelination.

Example 7 Anti-TIM-3-Mediated Activation and Proliferation of CD11b+Cells in Vitro

To further understand the function of TIM-3 in vivo and its observedrole in EAE, EAE-prone female SJL mice (4-8 weeks old) were injecteds.c. in each flank with 50 μg of PLP 139-151 emulsified in CFA andinjected i.p. every other day with either 100 μg anti-TIM-3 or 100 μgcontrol rIgG or PBS. Mice were sacrificed on day 10, and spleens wereremoved and spleen cells were isolated. The spleen cells were plated at5×10⁵ cells/well in round-bottom 96-well plates and stimulated for 48hrs with increasing concentrations (0-100 μg/mL) of PLP 139-151 orneuraminidase 101-120 peptide EALVRQGLAKVAYVYKPNNT (SEQ ID NO:12; Nase;Quality Controlled Biochemicals) as control antigen. Plates were thenpulsed with 1 μCi ³[H]-thymidine/per well for 12 hrs. The incorporatedradiolabeled thymidine was measured utilizing a Beta Plate scintillation

counter (Wallac). Whereas spleen cells from control rIgG2a-treated miceshowed a low basal (background) proliferative response (1000-5000 cpm)and a dose-dependent increase in proliferation with the addition ofspecific antigen, the spleen cells from anti-TIM-3-treated mice had 6-10times the basal response in the absence of antigen, although theresponse to specific antigen was similar (FIG. 5A). Flow cytometricanalysis of spleen cells from anti-TIM-3-treated mice revealed a 2-3fold increase in CD11b+ cell population, i.e., the population of cellsthat includes predominantly monocyte/macrophages.

To confirm whether CD11b+ cells in the anti-TIM-3-treated mice wereproliferating and might thus contribute to the basal proliferativeresponses, 5-bromodeoxyuridine (BrdU) was administered in the drinkingwater (8 mg/mL) of immunized SJL mice treated with anti-TIM-3 or rIgG2acontrol antibody as above. Mice were sacrificed on day 10 andsplenocytes were stained with mAbs to CD4, CD8, CD11b, CD11c, CD19, B220and BrdU. Flow cytometric analysis of the splenocytes fromanti-TIM-3-treated mice versus the splenocytes from rIgG2a-treated micerevealed an increase in the number of both BrdU+ and BrdU− cells onlyfor CD11b+ cells (FIG. 5B). Two thirds of these CD11b+ cellsincorporated BrdU and expressed higher levels of MHC class II (FIG. 5B),consistent with the view that these activated CD11b+ cells play a majorrole in the high basal response of anti-TIM-3 treated mice.

Example 8 Synergistic Effect of Anti-TIM-3-Treated APC Plus T Cells onBasal Proliferative Response

To determine the role of the T cells and the non-T cells, especially theCD11b+ cells, in the basal proliferative response, T cells and non-Tcells were purified from the spleen cells of the anti-TIM-3- andrIgG2a-treated mice. Either whole splenocytes or T cells (10⁵) wereincubated in the presence or absence of either 2×10⁵ B cells, 2×10⁵CD11b+ cells, or irradiated splenocytes, and the proliferative responsewas measured (³H-thymidine incorporation in triplicate wells). Whilethere was only a modest basal proliferative response from the purified Tcells, B cells or CD11b+ monocytes alone from the anti-TIM-3-treatedmice, the co-culturing of both B cells and CD11b+ cells with theanti-TIM-3-treated T cells recapitulated the high basal response (FIG.5C). In comparison, the addition of either B cells, monocytes or both Bcells and monocytes to the T cells from rIgG2a-treated mice producedonly an additive effect (FIG. 5C (black bars)). When crisscrossproliferation experiments were conducted, the addition ofanti-TIM-3-treated T cells to rIgG2a-treated B cells plus CD11b+ cellsincreased the basal proliferative response; however, the maximal basalproliferation required the presence of both anti-TIM-3-treated non-Tcells and anti-TIM-3-treated T cells. This basal proliferative responsewas not lowered by incubating the splenocytes with Fc receptor(FcR)-blocking antibodies, suggesting that FcR-mediated crosslinking canbe excluded as the mechanism. The synergistic effect of the interactionbetween T cells and non-T cells was further confirmed as the addition ofirradiated non-T cells to anti-TIM-3-treated T cells did notreconstitute the same high basal proliferative response.

Example 9 Cognate Interaction Between T Cells and Non-T Cells

To determine whether a cognate interaction was required between T cellsand non-T cells for the increase in the basal responses, in a separateset of experiments T cells and non-T cells were separated with apermeable 0.2 μm Anapore membrane that inhibits cell contact, andproliferative responses were measured. As also shown in FIG. 5C (graybars), separation of anti-TIM-3-treated T cells from anti-TIM-3-treatednon-T cells resulted in a dramatic decrease in the proliferativeresponses, indicating that a cognate interaction between T cells andnon-T cells is necessary.

Example 10 TIM-3-Expressing T Cells Regulate the Expansion andActivation of APCs

To address directly whether TIM-3-expressing T cells regulate theexpansion and activation of macrophages and other non-T cells, 5×10⁶TIM-3+ Th1 5B6 transgenic T cells (with specificity for PLP 139-151)were adoptively transferred to naïve SJL recipients, which were thenalso immunized with PLP 139-151 and treated with anti-TIM-3 or anti-ICOSantibody (as a T-cell antibody binding control) on days 0 and 2. Spleencells were removed on day 3 and the cells were stained with mAbs toCD11b, F4/80, B220 and MHC class II. Flow cytometric analysis of thespleen cells three days after transfer showed a dramatic increase in thenumber of activated CD11b+/F4/80+ macrophages in the anti-TIM-3-treatedgroup, but not in the anti-ICOS-treated mice. There was a 2-3 foldincrease in the number of CD11b+/F4/80+ cells present in subset 2 (FIG.5D). These F4/80+ macrophages also expressed higher levels of MHC classII, indicating that they were more activated.

Taken together, these data indicate that a cognate interaction betweennon-T cells and TIM-3-expressing Th1 cells is affected by anti-TIM-3treatment, resulting in the expansion and activation of CD11b+/F4/80+macrophages. Several possible mechanisms may explain this finding: a)Anti-TIM-3 may cross-link TIM-3 protein on the surface of differentiatedTh1 cells in vivo and amplify the production of pro-inflammatorycytokines (e.g., IFN-γ and TNF), which in turn may induce activation ofmacrophages; b) anti-TIM-3 antibody could enhance migration ofdifferentiated Th1 cells into the brain where these cells may increasethe cellular influx of macrophages from the circulation; c) anti-TIM-3could block a cognate interaction of TIM-3 with its potential inhibitoryligand on macrophages, thus leading to enhanced macrophage activation inthe presence of pro-inflammatory cytokines produced by Th1 cells. It isto be understood that these different possible mechanisms of action arenot necessarily mutually exclusive, nor are they to be understood toexclude any other mechanism of action.

Additionally, since Th1 cells and Th2 cells cross-regulate each other'sfunctions, expression of TIM-3 on Th1 cells and subsequent traffickingof TIM-3-bearing Th1 cells to target tissue sites is believed also tohave a role in the regulation of asthma and atopy. Thus TIM-3, inaddition to being a molecule that is selectively expressed on thesurface of Th1 cells, plays a functional role in macrophage activationand increased severity of an autoimmune disease. These data togetherwith the results in asthma-resistant mice suggest that TIM-3 gene familymembers play an important role in the regulation of autoimmunity andallergy.

Example 11 TIM-3 Promotes Trafficking of Th1 Cells to Target Tissues

In addition to its role in macrophage activation, TIM-3 has a role intrafficking of effector Th1 cells. More specifically, TIM-3 expressionpromotes trafficking of Th1 cells into sites of inflammation and intothe target tissues where they can mediate tissue injury andautoimmunity. Thus affecting the expression of TIM-3 on the surface ofTh1 cells or interaction of TIM-3 with its ligand might either inhibitor enhance the trafficking of the Th1 cells into the tissue sites tomediate immune response and inflammation.

The following experiment was performed to test the hypothesis thatexpression of TIM-3 promotes trafficking of differentiated Th1 cells totarget tissue. PLP 139-151 specific transgenic T cells derived from 5B6transgenic mice were activated and polarized under Th1 conditions. Allthe cells were tested for TIM-3 expression by FACS staining. Theactivated, polarized T cells were harvested 10 days after the finalactivation and stained with the dye 5,6-carboxyfluorescein diacetatesuccinimidyl ester (CFSE), which marks these cells and which dilutes asthe cells divide. In brief, the 5B6 Th1 cells were incubated at roomtemperature in PBS supplemented with 3 μM CFSE (Molecular Probes,Eugene, Oreg.) at 1×10⁷ cells/mL for 5-10 min. The cells weresubsequently washed in RPMI, resuspended in PBS, and transferred (1×10⁷cells/mouse) into naïve syngeneic recipients. The recipients were thenimmunized with the PLP 139-151 peptide in CFA and were also given 100 μgof anti-TIM-3 or control antibodies (anti-ICOS or anti-ICOSL) everyother day during the course of the experiment. The mice were sacrificedon day 3 and day 7, and the presence and number (percentage) ofCFSE-labeled cells were analyzed in the lymph nodes, spleens and brainsof the transferred mice by flow cytometry. On day 3 post-transfer, theCFSE-labeled cells were detected only in the spleens and not in thelymph nodes or brains of the transferred recipients (FIG. 6A). However,on day 7 in the group treated with anti-TIM-3 antibody, the vastmajority of CFSE-labeled cells were detected in the brains with adecrease in the number of CFSE-labeled cells in the spleen (FIG. 6B). Incontrast, in the control antibody-treated groups the vast majority ofthe CFSE-labeled cells were still detected in the spleens of these miceand very few cells were detected in the brains of these mice (FIG. 6B).The results of this experiment suggest that anti-TIM-3 antibodytreatment in vivo accelerates migration and trafficking of theTIM-3-expressing T cells into tissue sites including the CNS and thusenhances EAE induction.

Taken together, the data showing expression of TIM-3 on the majority ofthe infiltrating T cells in the CNS tissue and enhanced migration of thecells into the CNS following anti-TIM-3 treatment strongly support therole of TIM-3 in trafficking to the target tissue.

Example 12 TIM-3 Ligand is Expressed on APCs

To further elucidate the mechanism for activation of macrophages andCD11b+ cells by crosslinking of TIM-3 on Th1 cells, an experiment wasperformed to establish the presence of a ligand or receptor for TIM-3expressed on macrophages and other CD11b+ cell. A soluble TIM-3Ig fusionprotein was prepared, in which TIM-3 was genetically fused with humanimmunoglobulin constant region. This soluble protein was biotinylatedand used in flow cytometry experiments to identify the ligand for TIM-3on various cell types. As shown in FIG. 7, using this reagent it wasobserved that soluble TIM-3Ig bound to cells of a dendritic cell (D2Sc1)cell line and macrophage (RAW 264.7) cell line, but only weakly, if atall, to cells of a mouse B cell line (LS 102.9). Additional studiesdemonstrated TIM-3Ig bound to normal mouse cells in vitro and to CD11b+cells in vivo.

Example 13 Soluble TIM-3

Full-length and alternatively spliced forms of murine TIM-3 wereisolated using a RT-PCR based approach. Primers were designed in the 5′and 3′ untranslated region (UTR) of the murine TIM-3 gene, and subjectedto PCR using cDNA generated from concanavalin A activated splenocytes.Two amplicons, of approximately 1 kb and 800 bp in size, were cloned andsequenced. The predicted amino acid translation of the 1 kb amplicondemonstrated an open reading frame consisting of a signal peptide, IgV,mucin, transmembrane, and cytoplasmic domains. In contrast, analysis ofthe open reading frame from the 800 bp product demonstrated the presenceof only the signal peptide, IgV, and cytoplasmic domains. A deducedamino acid sequence for the alternatively spliced, soluble form of TIM-3is provided as SEQ ID NO:13.

Amino acid sequence of alternatively spliced form of TIM-3 SEQ ID NO: 13MFSGLTLNCV LLLLQLLLAR SLEDGYKVEV GKNAYLPCSY TLPTSGTLV PMCWGKGFCPW 60SQCTNELLRT DERNVTYQKS SRYQLKGDLN KGDVSLIIKN VTLDDHGTY CCRIQFPGLMN 120DKKLELKLDI KAGYSCKKKK LSSLSLITLA NLPPGGLANA GAVRIRSEE NIYTIEENVYE 180VENSNEYYCY VNSQQPS 197

Absence of the mucin domain and transmembrane region was consistent withsplicing of exon 3, exon 4, and exon 5 from the murine TIM-3 gene. Thedata is consistent with the product encoded by the 800 bp ampliconcorresponding to an alternatively spliced, soluble form of TIM-3.

Example 14 Soluble TIM-3 Fusion Protein Construction

Two fusion proteins containing extracellular portions of mouse TIM-3fused to a human Fc tail were constructed to identify potential TIM-3ligand(s) and to determine the functional role of TIM-3 ligand inregulating T-cell responses. One construct, mTIM-3/hFc fusion protein,contains the entire extracellular portion of mouse TIM-3 (IgV and mucindomains) fused to a human Fc tail. A second construct, mTIM-3Ig/hFcfusion protein, contains only the IgV domain of mouse TIM-3 fused to ahuman Fc tail. This second fusion protein was constructed as the datasuggesting the alternatively spliced form of TIM-3 containing only thesignal peptide, IgV, and cytoplasmic domains suggests that this secretedform of TIM-3 may have an Ig-specific ligand. These two fusion proteins,being TIM-3 ligand-binding molecules, can be used to identify andinteract with TIM-3 ligand.

Example 15 Hyperacute EAE Following Administration of Soluble TIM-3

To determine the effect of these soluble TIM-3 fusion protein constructson the development of experimental autoimmune encephalomyelitis (EAE),SJL mice were immunized with 50 μg PLP 139-151 in complete Freund'sadjuvant (CFA) plus 100 ng of pertussis toxin intravenously and treatedwith intraperitoneal administration 100 μg of mTIM-3/hFc or 100 μg ofmTIM-3Ig/hFc or 100 μg of human IgG (control) or 100 μl PBS every otherday from day 0-10. Mice treated with either mTIM-3Ig/hFc or mTIM-3/hFcdeveloped a more severe form of EAE, as demonstrated by both diseaseseverity and mortality (see Table 2). Mice treated with mTIM-3/hFc hadan average disease score of 2.69 versus an average disease score of 1.86and 1.89 for hIgG- and PBS-treated controls, respectively. Mice treatedwith mTIM-3/hFc also exhibited increased mortality—31.3% versus 5.8% and5.6% for hIgG- and PBS-treated controls, respectively. This increasedseverity and mortality upon treatment with mTIM-3/hFc is reminiscent ofthe increased EAE seen in mice treated with anti-TIM-3 antibody,suggesting that either blocking or activating the TIM-3 ligand causesthe same effect as blocking or activating TIM-3 itself

TABLE 2 EAE in Mice Treated with Soluble TIM-3 or Control Mean DayTreatment Incidence Mortality of Onset Mean Score mTIM-3/hFc 16/16 2/1612.3 2.69 (100%)   (13%) hIgG 16/17 1/17 13.8 1.86 (94%) (5.8%) PBS17/18 1/18 12.9 1.89 (94%) (5.6%)

Example 16 Soluble TIM-3-Mediated Activation and Proliferation of SpleenCells

To better understand the in vivo mechanism that leads to this increasedEAE, SJL mice that were immunized and treated with fusion protein orcontrol as described in Example 15 were sacrificed on day 10, andspleens were removed. Whole spleen cells were plated at 5×10⁵ cells/wellfor a proliferation assay measured by thymidine incorporation. Whilebasal proliferation for hIgG- and PBS-treated controls was low(approximately 10,000 cpm), spleen cells from mice treated withmTIM-3/hFc (approximately 60,000 cpm) or mTIM-3Ig/hFc (approximately140,000 cpm) proliferated at very high levels without peptiderestimulation (FIG. 8A). This result is again reminiscent of that seenwhen mice were treated with anti-TIM-3 antibody. Cytokine production wasmeasured by sandwich ELISA on supernatants taken at 48 h. Mice treatedwith mTIM-3/hFc produced high levels of IFN-γ (4111 pg/ml) and IL-2 (592pg/ml), while hIgG- and PBS-treated controls did not produce any IFN-γor IL-2 (FIG. 8B). Mice treated with mTIM-3/hFc also produced threetimes more IL-4 than hIgG-treated controls (FIG. 8B). Taken together,this data demonstrates that whole spleen cells taken from treated micewith either mTIM-3Ig/hFc or mTIM-3/hFc are highly activated and producelarge amounts of Th1 cytokines (IFN-γ and IL-2) as well as significantamounts of Th2 cytokine IL-4. No IL-10 or TNF-α was detected.

To see what types of cells compose the activated spleen milieu ofmTIM-3/hFc-treated mice, whole spleen cells taken on day 10 afterimmunization and fusion protein treatment were stained directly ex vivoand subjected to FACS analysis. CD4+ and CD8+ T cells from spleens ofmice treated with mTIM-3/hFc expressed significantly higher levels ofactivation markers CD25 and CD69 than did corresponding cells from micetreated with negative control hIgG or PBS. This data indicated that Tcells from spleens of immunized SJL mice treated with mTIM-3/hFc arehighly activated without peptide restimulation.

Example 17 Use of Soluble TIM-3 to Identify TIM-3 Ligand

To determine what cell(s) may express the TIM-3 ligand(s), themTIM-3/hFc and mTIM-3Ig/hFc fusion proteins were used for staining naïvespleen cells from SJL, NOD, BALB/c and C57B L/6 mice. Individualpopulations of spleen cells were identified by co-staining withappropriately selected cell surface markers. Flow cytometric analysisrevealed that mTIM-3Ig/hFc specifically stained naïve and activated CD4+T cells. Thus one potential ligand for the alternatively spliced,soluble form of TIM-3 appeared to be expressed on CD4+ T cells.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations. Various equivalents, changes and modifications may be madewithout departing from the spirit and scope of this invention, and it isunderstood that such equivalent embodiments are part of this invention.All of the references, patents, and patent publications identified orcited herein are incorporated, in their entirety, by reference.

We claim:
 1. A method for treating a subject having cancer, the methodcomprising administering a TIM-3 (T cell Immunoglobulin and Mucin domaincontaining molecule-3) binding molecule in an amount effective topromote T-cell trafficking to a target tissue, wherein the TIM-3 bindingmolecule is a TIM-3 antibody or antigen-binding fragment thereof, incombination with a chemotherapeutic agent, thereby treating the cancer.2. The method of claim 1, wherein the chemotherapeutic agent is directlytoxic to cancer cells.
 3. The method of claim 2, wherein thechemotherapeutic agent is an alkylating/alkaloid agent.
 4. The method ofclaim 3, wherein the alkylating/alkaloid agent comprises a nitrogenmustard.
 5. The method of claim 4, wherein the nitrogen mustard isselected from the group consisting of: mechlorethamin HCl, chlorambucil,and ifosfamide.
 6. The method of claim 3, wherein thealkylating/alkaloid agent is selected from the group consisting of:cisplatin, carboplatin, carmustine, lomustine, streptozocin, busulfan,procarbozine, and temozolomide.
 7. The method of claim 2, wherein thechemotherapeutic agent is selected from the group consisting of:busulfan, capecitabine, carboplatin, carmustaine, chlorambucil,cisplatin, cladribine, cytarabine HCl, daunorubicine HCl, doxorubicin,epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine,ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine HCl (nitrogenmustard), mercaptopurine, mitoxantrone, mitoxantrone HCl, paclitaxel,pentostatin, plicamycin, semustine, streptozocin, taxane analog,temozolomide, teniposide (VM-26), thioguanine, vinblastine sulfate, andvindesine sulfate.
 8. The method of claim 1, wherein the TIM-3 antibodyor antigen-binding fragment thereof binds to an extracellular region ofTIM-3.
 9. The method of claim 1, wherein the TIM-3 antibody orantigen-binding fragment thereof is administered directly to the targettissue.
 10. The method of claim 1, wherein the TIM-3 antibody orantigen-binding fragment thereof is administered intravenously.